Conformational restriction of cyanine fluorophores in far-red and near-ir range

ABSTRACT

Conformationally restricted cyanine fluorophores, as well as methods of making and using the compounds, are described. The conformationally restricted cyanine fluorophores have a chemical structure according to Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof: 
     
       
         
         
             
             
         
       
     
     wherein A is 
     
       
         
         
             
             
         
       
     
     and wherein each “*” designates an attachment point of A.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. provisional patent applicationNo. 62/549,566 filed Aug. 24, 2017, which is incorporated by referenceherein in its entirety.

FIELD

Conformationally restricted cyanine fluorophores are disclosed, andmethods of making and using the conformationally restricted cyaninefluorophores.

BACKGROUND

Single molecule localization microscopy (SMLM) techniques likephotoactivated localization microscopy (PALM) and direct stochasticoptical reconstruction microscopy (dSTORM) enable three-dimensional (3D)imaging of cellular components with nearly molecular resolution.Localization precision, and therefore the structural resolutioncapability of SMLM, scales with the inverse square root of the singlemolecule emitter intensity. Consequently, SMLM fluorophores shouldprovide high photon yields in the on state, while exhibiting low toabsent background fluorescence in the off state.

Indocyanines are among the most useful fluorescent small molecules,uniquely spanning the visible to near-infrared (near-IR) range throughsuccessive 2-carbon homologation. Far-red variants, including Cy5 andAlexa 647, are the most common chemical component of dSTORM methods.However, fluorescence quantum yields (Φ_(F)) are modest, typically below0.2 in aqueous solution. Cyanine excited state deactivation involvestrans- to cis-polyene rotation that competes extensively with photonemission. In the trimethine series, which emits in the green region ofthe spectrum, this pathway has been obstructed through a syntheticstrategy involving installation of fused 6-membered rings along thepolymethine bridge dramatically improving quantum yield. However, thesynthetic strategy is not applicable to far-red cyanines, which requiresynthesis of a complex fused tetracyclic or pentacyclic ring system.

SUMMARY

Embodiments of conformationally restricted cyanine fluorophores, as wellas methods of making and using the conformationally restricted cyaninefluorophores, are disclosed. Embodiments of the disclosed compounds havea chemical structure according to Formula I, or a stereoisomer orpharmaceutically acceptable salt thereof:

wherein A is

wherein each “*” designates an attachment point of A; the bondsrepresented by “

” are single or double bonds as needed to satisfy valence requirements;R¹-R⁹ and R¹¹ independently are H, deuterium, alkyl, heteroalkyl,—N(R^(a))₂, sulfonate, alkyl sulfonate, amino, aminoalkyl, —C(O)OR^(a),trityl, or a group comprising a conjugatable moiety, a targeting agent,or a drug, where each R^(a) independently is H, deuterium, alkyl orheteroalkyl; R¹⁰ is H, deuterium, O, alkyl, aryl, amino, sulfonate,triflate, —C(O)OR^(b), —OR^(b), —N(R^(b))₂, heteroalkyl, heteroaryl,trityl, or a group comprising a conjugatable moiety, a targeting agent,or a drug, where each R^(b) independently is H, deuterium, alkyl,heteroalkyl, aryl, or heteroaryl; and Y¹ and Y² independently areC(R^(c))₂, N(R^(d)), S, O, or Se, wherein each R^(c) independently is H,deuterium, alkyl, —(OCH₂CH₂)_(x)OH where x is an integer ≥2, trityl, ora group comprising a conjugatable moiety, a targeting agent, or a drug,and each R^(d) independently is H, deuterium, alkyl, or heteroalkyl.

In some embodiments, the compound has a chemical structure according toFormula IA, IB, IC, or ID:

In certain embodiments, the compound has a chemical structure accordingto Formula II or Formula III:

Embodiments of a pharmaceutical conformation comprise a compoundaccording to Formula I and a pharmaceutically acceptable carrier.

Embodiments of a method for making a conformationally restrictedpentamethine cyanine compound according to Formula I proceed via acyclization cascade of a dialdehyde precursor, which is accessed throughchemoselective olefin metathesis. The dialdehyde undergoesintramolecular Michael addition followed by a dihydropyran ring-formingcascade. Embodiments of a method for making a conformationallyrestricted heptamethine cyanine compound according to Formula I includecombining an indolene precursor or two distinct indolenine precursorswith a bis vinylogous amide to provide a heptamethine cyanine includingpendant terminal alkenyl groups, which are subsequently converted todialdehydes. A cyclization reaction produces the conformationallyrestricted heptamethine cyanine compound.

Embodiments of compounds according to Formula I may be used for imagingapplications. Some embodiments of a method for using a compoundaccording to Formula I, wherein the compound includes a target agent,include combining the compound with a sample comprising a target capableof binding with the targeting agent; and imaging the target byvisualizing the compound. In some embodiments, visualizing the compoundincludes irradiating the sample with targeted application of a quantityof light having a wavelength in the visible or near-infrared range and aselected intensity, wherein the quantity of light is sufficient toproduce fluorescence of the compound; and detecting any fluorescenceemitted by the compound. In any or all of the above embodiments,combining the compound with the sample may be performed in vitro, exvivo, or in vivo. In any or all of the above embodiments, the method mayfurther include combining the compound with a reducing agent prior toimaging the target. In any or all of the above embodiments, the samplemay be a tissue sample, a biological fluid, or a target area within asubject. In some embodiments, the sample is a target area within asubject, and the method further includes administering the compound, ora pharmaceutical composition comprising the compound to the subject,subsequently irradiating the compound by targeted application of thequantity of light to a targeted portion of the subject, and detectingany fluorescence from the compound in the targeted portion of thesubject. In certain embodiments, the target area is a tumor site, thetargeted portion of the subject includes the tumor site, and the methodfurther includes excising at least a portion of the tumor from thesubject after detecting the fluorescence in the targeted portion of thesubject.

In an independent embodiment, a method for detective reactive oxygenspecies includes combining a compound according to Formula I with areducing agent to provide a reduced compound; contacting a sample withthe reduced compound, whereby the reduced compound is oxidized toregenerate the compound according to Formula I if reactive oxygenspecies (ROS) are present in the sample; irradiating the sample with aquantity of light having a wavelength in the visible or near-infraredrange and a selected intensity, wherein the quantity of light issufficient to produce fluorescence if the reduced compound has beenoxidized by the ROS to regenerate the compound according to Formula I;and detecting any fluorescence emitted by the compound according toFormula I, wherein fluorescence indicates the presence of ROS in thesample.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exemplary retrosynthetic pathway for making aconformationally restricted pentamethine cyanine fluorophore.

FIG. 2 is an exemplary synthetic scheme for preparing an unsubstituted,conformationally restricted pentamethine cyanine fluorophore.

FIG. 3 is an exemplary synthetic scheme for preparing a conjugatable,conformationally restricted pentamethine cyanine fluorophore.

FIG. 4 shows a chemical structure and NMR analysis of an exemplaryconformationally restricted pentamethine cyanine fluorophore (compound4).

FIG. 5 shows diastereomers of compound 4 and their associated energies.

FIG. 6 shows a chemical structure and NMR analysis of an exemplaryconformationally restricted, phalloidin-conjugated pentamethine cyaninefluorophore (compound 7).

FIG. 7 is a graph showing the temperature dependence of fluorescenceemission from compounds 4 (squares) and 10 (circles) in ethanol.

FIG. 8 is a graph showing ultraviolet recovery of 20 μM solutions ofcompound 4 (squares) and 10 (circles) in 4:1 PBS (50 mM, pH 7.4):DMSOfollowing NaBH₄ reduction (2.5 mM in 1:1 DMSO:MeOH). Absorbance wasmeasured at λ_(max) (compound 4 at 640 nm and compound 10 at 660 nm as afunction of time of 365 nm irradiation (5 mW/cm²).

FIG. 9 is a graph showing the effect of TCEP addition on absorbance ofcompound 7 and AF647 (10 μM each). Absorbance at λ_(max) (compound 7 at670 nm and AF647 at 650 nm) as a function of TCEP concentration in Tris(0.20 M, pH 9.0).

FIG. 10 is a color 3D TRABI_BP SMLM image of F-actin in a U2OS (humanbone osteosarcoma epithelial) cell with compound 9, a conformationallyrestricted phalloidin-pentamethine cyanine fluorophore conjugate.

FIG. 11 shows lateral and axial localization precisions calculated fromthe image of FIG. 10. Marks indicate data points and solid linesindicate Gaussian fits to the data.

FIG. 12 is a bar graph showing a comparison of single molecule photonintensities regarding single frame (black) and tracked (grey) medianvalues of data illustrated for compound 9_(3D) (left) and comparablemeasurements in 2D imaging modes of compound 9 (center) and AF647(right) in standard dSTORM photoswitching buffer.

FIG. 13 is a bar graph showing experimentally determined laterallocalization precisions of compound 9_(3D) (6.9 nm), compound 9_(2D)(5.2 nm) and AF6472D (5.9 nm).

FIG. 14 is a schematic diagram illustrating one embodiment of a methodfor using the disclosed compounds according to Formula I by injection ofthe compound followed by targeted delivery of light of a desiredwavelength to the external surface of the skin.

FIG. 15 is an exemplary synthetic scheme for preparing a bis-sulfonated,conformationally restricted pentamethine cyanine fluorophore.

DETAILED DESCRIPTION

This disclosure concerns embodiments of conformationally restrictedcyanine fluorophores, and methods of making and using theconformationally restricted cyanine fluorophores. Advantageously, theconformationally restricted cyanine fluorophores have emission maximathat are in the far-red and near-infrared range, and/or have absorptionand/or emission maxima that are red-shifted compared to correspondingcyanine fluorophores that are not conformationally restricted.

I. Definitions and Abbreviations

The following explanations of terms and abbreviations are provided tobetter describe the present disclosure and to guide those of ordinaryskill in the art in the practice of the present disclosure. As usedherein, “comprising” means “including” and the singular forms “a” or“an” or “the” include plural references unless the context clearlydictates otherwise. The term “or” refers to a single element of statedalternative elements or a combination of two or more elements, unlessthe context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features of thedisclosure are apparent from the following detailed description and theclaims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, percentages, temperatures, times, and soforth, as used in the specification or claims are to be understood asbeing modified by the term “about.” Accordingly, unless otherwiseindicated, implicitly or explicitly, the numerical parameters set forthare approximations that may depend on the desired properties soughtand/or limits of detection under standard test conditions/methods. Whendirectly and explicitly distinguishing embodiments from discussed priorart, the embodiment numbers are not approximates unless the word “about”is recited.

Definitions of common terms in chemistry may be found in Richard J.Lewis, Sr. (ed.), Hawley's Condensed Chemical Dictionary, published byJohn Wiley & Sons, Inc., 1997 (ISBN 0-471-29205-2). Definitions ofcommon terms in molecular biology may be found in Benjamin Lewin, GenesVII, published by Oxford University Press, 2000 (ISBN 019879276X);Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, publishedby Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by Wiley, John & Sons, Inc., 1995 (ISBN0471186341); and other similar references.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Aliphatic: A substantially hydrocarbon-based compound, or a radicalthereof (e.g., C₆H₁₃, for a hexane radical), including alkanes, alkenes,alkynes, including cyclic versions thereof, and further includingstraight- and branched-chain arrangements, and all stereo and positionisomers as well. Unless expressly stated otherwise, an aliphatic groupcontains from one to twenty-five carbon atoms; for example, from one tofifteen, from one to ten, from one to six, or from one to four carbonatoms. The term “lower aliphatic” refers to an aliphatic groupcontaining from one to ten carbon atoms. An aliphatic chain may besubstituted or unsubstituted. Unless expressly referred to as an“unsubstituted aliphatic,” an aliphatic group can either beunsubstituted or substituted. An aliphatic group can be substituted withone or more substituents (up to two substituents for each methylenecarbon in an aliphatic chain, or up to one substituent for each carbonof a —C═C— double bond in an aliphatic chain, or up to one substituentfor a carbon of a terminal methine group). Exemplary substituentsinclude, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy,alkylamino, alkylthio, acyl, aldehyde, amide, amino, aminoalkyl, aryl,arylalkyl, carboxyl, cyano, cycloalkyl, dialkylamino, halo,haloaliphatic, heteroaliphatic, heteroaryl, heterocycloaliphatic,hydroxyl, oxo, sulfonamide, sulfhydryl, thioalkoxy, or otherfunctionality.

Alkoxy: A group having the structure —OR, where R is a substituted orunsubstituted alkyl. Methoxy (—OCH₃) is an exemplary alkoxy group. In asubstituted alkoxy, R is alkyl substituted with a non-interferingsubstituent.

Alkoxy carbonyl: A group having the structure —(O)C—O—R, where R is asubstituted or unsubstituted alkyl.

Alkyl: A hydrocarbon group having a saturated carbon chain. The chainmay be branched, unbranched, or cyclic (cycloalkyl). The term loweralkyl means the chain includes 1-10 carbon atoms. Unless otherwisespecified, the term alkyl encompasses substituted and unsubstitutedalkyl.

Alkyl sulfonate: A group having the structure —R—SO₃ ⁻, where R is asubstituted or unsubstituted alkyl.

Amino: A group having the structure —N(R)R′ where R and R′ areindependently hydrogen, haloalkyl, aliphatic, heteroaliphatic, aryl(such as optionally substituted phenyl or benzyl), heteroaryl,alkylsulfano, or other functionality. A “primary amino” group is —NH₂.“Mono-substituted amino” means a radical —N(H)R substituted as above andincludes, e.g., methylamino, (1-methylethyl)amino, phenylamino, and thelike. “Di-substituted amino” means a radical —N(R)R′ substituted asabove and includes, e.g., dimethylamino, methylethylamino,di(1-methylethyl)amino, and the like. The term amino also encompassescharged tri-substituted amino groups, e.g., —N(R)(R′)R″⁺ where R, R′,and R″ are independently hydrogen, haloalkyl, aliphatic,heteroaliphatic, aryl (such as optionally substituted phenyl or benzyl),heteroaryl, alkylsulfano, or other functionality.

Aminoalkyl: A chemical functional group —RNH₂ or —RNH₃ ⁺ where R is analkyl group. “Substituted aminoalkyl” means that the amino group issubstituted, e.g., —RN(R′)R″ or —RN(R′)(R″)R′″⁺ where R′, R″, and R′″are independently hydrogen, haloalkyl, aliphatic, heteroaliphatic, aryl(such as optionally substituted phenyl or benzyl), heteroaryl,alkylsulfano, or other functionality.

Antibody: A protein (or protein complex) that includes one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad of immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Inavian and reptilian species, IgY antibodies are equivalent to mammalianIgG.

The basic immunoglobulin (antibody) structural unit is generally atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about kDa) and one“heavy” (about 50-70 kDa) chain. The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(V_(L)) and “variable heavy chain” (V_(H)) refer, respectively, to theselight and heavy chains.

The structure of IgY antibodies is similar to the structure of mammalianIgG, with two heavy (“nu” chains; approximately 67-70 kDa) and two lightchains (22-30 kDa). The molecular weight of an IgY molecule is about 180kDa, but it often runs as a smear on gels due to the presence of about3% carbohydrate. Heavy chains (H) of IgY antibodies are composed of fourconstant domains and one variable domain, which contains theantigen-binding site.

As used herein, the term “antibodies” includes intact immunoglobulins aswell as a number of well-characterized fragments. For instance, Fabs,Fvs, and single-chain Fvs (SCFvs) that bind to target protein (orepitope within a protein or fusion protein) would also be specificbinding agents for that protein (or epitope). These antibody fragmentsare defined as follows: (1) Fab, the fragment which contains amonovalent antigen-binding fragment of an antibody molecule produced bydigestion of whole antibody with the enzyme papain to yield an intactlight chain and a portion of one heavy chain; (2) Fab′, the fragment ofan antibody molecule obtained by treating whole antibody with pepsin,followed by reduction, to yield an intact light chain and a portion ofthe heavy chain; two Fab′ fragments are obtained per antibody molecule;(3) (Fab′)₂, the fragment of the antibody obtained by treating wholeantibody with the enzyme pepsin without subsequent reduction; (4)F(ab′)₂, a dimer of two Fab′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody, a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule. Methods of makingthese fragments are routine (see, for example, Harlow and Lane, UsingAntibodies: A Laboratory Manual, CSHL, New York, 1999). As used herein,the term “antibodies” includes antibodies comprising one or moreunnatural (i.e., non-naturally occurring) amino acids (e.g.,p-acetyl-phenylalanine) to facilitate site-specific conjugation.

Antibodies for use in the methods of this disclosure can be monoclonalor polyclonal, and for example specifically bind a target such as thetarget antigen. Merely by way of example, monoclonal antibodies can beprepared from murine hybridomas according to the classical method ofKohler and Milstein (Nature 256:495-97, 1975) or derivative methodsthereof. Detailed procedures for monoclonal antibody production aredescribed in Harlow and Lane, Using Antibodies: A Laboratory Manual,CSHL, New York, 1999.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. As used herein, a“target antigen” is an antigen (including an epitope of the antigen)that is recognized and bound by a targeting agent. “Specific binding”does not require exclusive binding. In some embodiments, the antigen isobtained from a cell or tissue extract. In some embodiments, the targetantigen is an antigen on a tumor cell. An antigen need not be afull-length protein. Antigens contemplated for use include anyimmunogenic fragments of a protein, such as any antigens having at leastone epitope that can be specifically bound by an antibody.

Aryl: A monovalent aromatic carbocyclic group of, unless specifiedotherwise, from 6 to carbon atoms having a single ring (e.g., phenyl) ormultiple condensed rings in which at least one ring is aromatic (e.g.,quinoline, indole, benzodioxole, and the like), provided that the pointof attachment is through an atom of an aromatic portion of the arylgroup and the aromatic portion at the point of attachment contains onlycarbons in the aromatic ring. If any aromatic ring portion contains aheteroatom, the group is a heteroaryl and not an aryl. Aryl groups aremonocyclic, bicyclic, tricyclic or tetracyclic. Unless otherwisespecified, the term aryl encompasses substituted and unsubstituted aryl.

Biological sample: As used herein, a “biological sample” refers to asample obtained from a subject (such as a human or veterinary subject)or other type of organism, such as a plant, bacteria or insect.Biological samples from a subject include, but are not limited to,cells, tissue, serum, blood, plasma, urine, saliva, cerebral spinalfluid (CSF) or other bodily fluid. In particular examples of the methoddisclosed herein, the biological sample is a tissue sample.

Conformationally restricted: The term “conformationally restricted” asused herein refers to a cyanine compound that has been modified so thatthe molecule loses flexibility in the region of the central conjugatedpolymethine bridge. The term “rigidized” is a synonym for“conformationally restricted.”

Conjugatable moiety: A portion of a molecule that allows the molecule tobe conjugated (i.e., coupled or bound) to another molecule, e.g., to adrug or targeting agent such as an antibody.

dSTORM: Direct stochastic optical reconstruction microscopy.

Drug: As used herein, the term “drug” refers to a substance which has aphysiological effect when administered to a subject, and is intended foruse in the treatment, mitigation, cure, prevention, or diagnosis ofdisease or used to otherwise enhance physical or mental well-being.

The term “small molecule drug” refers to a drug having a molecularweight <1,000 Daltons.

An anti-cancer drug is a drug that is used to treat malignancies.Exemplary anti-cancer drugs include, but are not limited to,abiraterone, actinomycin D, altretamine, amifostine, anastrozole,asparaginase, bexarotene, bicalutamide, bleomycin, buserelin, busulfan,carboplatin, carmustine, chlorambucil cisplatin, cladribine, clodronate,combretastatin A4, cyclophosphamide, cyproterone, cytarabine,dacarbazine, daunorubicin, degarelix, diethylstilbestrol, docetaxel,doxorubicin, duocarmycin DM, epirubicin, ethinyl estradiol, etoposide,exemestane, 5-fluorouracil, fludarabine, flutamide, folinic acid,fulvestrant, gemcitabine, goserelin, ibandronic acid, idarubicin,ifosfamide, irinotecan, lanreotide, lenalidomide, letrozole,leuprorelin, medroxyprogesterone, megestrol, melphalan, mesna,methotrexate, octreotide, pamidronate, pemetrexed, mitocmycin, mitotane,mitoxantrone, oxaliplatin, paclitaxel, pentastatin, pipbroman,plicamycin, procarbazine, raltitrexed, stilbestrol, streptozocin,tamoxifen, temozolomide, teniposide, topotecan, triptorelin,vinblastine, vincristine, vinorelbine, and zolendronic acid.

Effective amount or therapeutically effective amount: An amountsufficient to provide a beneficial, or therapeutic, effect to a subjector a given percentage of subjects.

Epitope: An antigenic determinant. Epitopes are particular chemicalgroups or contiguous or non-contiguous peptide sequences on a moleculethat are antigenic, that is, that elicit a specific immune response. Anantibody binds a particular antigenic epitope based on the threedimensional structure of the antibody and the matching (or cognate)epitope.

Far-red: Far red light is generally considered to be light is awavelength within a range of 700-850 nm.

Heteroaliphatic: An aliphatic compound or group having at least oneheteroatom, i.e., one or more carbon atoms has been replaced with anatom having at least one lone pair of electrons, typically nitrogen,oxygen, phosphorus, silicon, or sulfur. Heteroaliphatic compounds orgroups may be substituted or unsubstituted, branched or unbranched,cyclic or acyclic, and include “heterocycle”, “heterocyclyl”,“heterocycloaliphatic”, or “heterocyclic” groups.

Heteroalkyl: An alkyl group as defined above containing at least oneheteroatom, such as N, O, S, or S(O)_(n) (where n is 1 or 2). Unlessotherwise specified, the term heteroalkyl encompasses substituted andunsubstituted heteroalkyl.

Heteroaryl: An aromatic compound or group having at least oneheteroatom, i.e., one or more carbon atoms in the ring has been replacedwith an atom having at least one lone pair of electrons, typicallynitrogen, oxygen, phosphorus, silicon, or sulfur. Unless otherwisespecified, the term heteroaryl encompasses substituted and unsubstitutedheteroaryl.

Ligand: A molecule that binds to a receptor, having a biological effect.

Linker: A molecule or group of atoms positioned between two moieties. Asused herein, the term “linker” refers to a group of atoms positionedbetween the cyanine fluorophore and a targeting agent or reactive group,or to a group of atoms positioned between the cyanine fluorophore and adrug.

Near-infrared (near-IR, NIR): Wavelengths within the range of 650-2500nm. Unless otherwise specified, the terms “near-infrared” and “NIR” asused herein refer to wavelengths within the range of 650-900 nm.

PALM: Photo-activated localization microscopy.

PBS: Phosphate-buffered saline.

Pharmaceutically acceptable carrier: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington: The Science and Practice of Pharmacy, The University of theSciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins,Philadelphia, Pa., 21′ Edition (2005), describes compositions andformulations suitable for pharmaceutical delivery of one or moreconformationally restricted cyanine fluorophores as disclosed herein.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. In some examples, the pharmaceutically acceptable carrier maybe sterile to be suitable for administration to a subject (for example,by parenteral, intramuscular, or subcutaneous injection). In addition tobiologically-neutral carriers, pharmaceutical compositions to beadministered can contain minor amounts of non-toxic auxiliarysubstances, such as wetting or emulsifying agents, preservatives, and pHbuffering agents and the like, for example sodium acetate or sorbitanmonolaurate.

Pharmaceutically acceptable salt: A biologically compatible salt of adisclosed conformationally restricted cyanine fluorophores, which saltsare derived from a variety of organic and inorganic counter ions wellknown in the art and include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium, and the like; and whenthe molecule contains a basic functionality, salts of organic orinorganic acids, such as hydrochloride, hydrobromide, tartrate,mesylate, acetate, maleate, oxalate, and the like. Pharmaceuticallyacceptable acid addition salts are those salts that retain thebiological effectiveness of the free bases while formed by acid partnersthat are not biologically or otherwise undesirable, e.g., inorganicacids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitricacid, phosphoric acid, and the like, as well as organic acids such asacetic acid, trifluoroacetic acid, propionic acid, glycolic acid,pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like. Pharmaceuticallyacceptable base addition salts include those derived from inorganicbases such as sodium, potassium, lithium, ammonium, calcium, magnesium,iron, zinc, copper, manganese, aluminum salts and the like. Exemplarysalts are the ammonium, potassium, sodium, calcium, and magnesium salts.Salts derived from pharmaceutically acceptable organic non-toxic basesinclude, but are not limited to, salts of primary, secondary, andtertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins, and the like. Exemplaryorganic bases are isopropylamine, diethylamine, ethanolamine,trimethylamine, dicyclohexylamine, choline, and caffeine. (See, forexample, S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm. Sci.,1977; 66:1-19, which is incorporated herein by reference.)

Phosphoramidite: A group having the general formula (RO)₂PNR₂. As asubstituent, a phosphoramidite has a general formula —RO—P(OR)NR₂ whereeach R independently is aliphatic, such as substituted or unsubstitutedalkyl.

Protecting group: When synthesizing organic compounds, often a specificfunctional group cannot survive the required reagents or chemicalenvironments. These groups must be protected. A protecting group, orprotective group, is introduced into a molecule by chemical modificationof a functional group in order to obtain chemoselectivity in asubsequent chemical reaction. Various exemplary protecting or protectivegroups are disclosed in Greene's Protective Groups in Organic Synthesis,by Peter G. M. Wuts and Theodora W. Greene (Oct. 30, 2006), which isincorporated herein by reference.

SMLM: Single-molecule localization microscopy.

Specific binding partner: A member of a pair of molecules that interactby means of specific, non-covalent interactions that depend on thethree-dimensional structures of the molecules involved. Exemplary pairsof specific binding partners include antigen/antibody, hapten/antibody,receptor/ligand, nucleic acid strand/complementary nucleic acid strand,substrate/enzyme, inhibitor/enzyme, carbohydratelectin, biotin/avidin(such as biotin/streptavidin), and virus/cellular receptor.

STORM: Stochastic optical reconstruction microscopy.

Substituent: An atom or group of atoms that replaces another atom in amolecule as the result of a reaction. The term “substituent” typicallyrefers to an atom or group of atoms that replaces a hydrogen atom, ortwo hydrogen atoms if the substituent is attached via a double bond, ona parent hydrocarbon chain or ring. The term “substituent” may alsocover groups of atoms having multiple points of attachment to themolecule, e.g., the substituent replaces two or more hydrogen atoms on aparent hydrocarbon chain or ring. In such instances, the substituent,unless otherwise specified, may be attached in any spatial orientationto the parent hydrocarbon chain or ring. Exemplary substituents include,for instance, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio,acyl, aldehyde, amido, amino, aminoalkyl, aryl, arylalkyl, arylamino,carbonate, carboxyl, cyano, cycloalkyl, dialkylamino, halo,haloaliphatic (e.g., haloalkyl), haloalkoxy, heteroaliphatic,heteroaryl, heterocycloaliphatic, hydroxyl, isocyano, isothiocyano, oxo,sulfonamide, sulfhydryl, thio, and thioalkoxy groups.

Substituted: A fundamental compound, such as an aryl or aliphaticcompound, or a radical thereof, having coupled thereto one or moresubstituents, each substituent typically replacing a hydrogen atom onthe fundamental compound. Solely by way of example and withoutlimitation, a substituted aryl compound may have an aliphatic groupcoupled to the closed ring of the aryl base, such as with toluene. Againsolely by way of example and without limitation, a long-chainhydrocarbon may have a hydroxyl group bonded thereto.

Sulfonate-containing group: A group including SO₃ ⁻. The termsulfonate-containing group includes —SO₃ and —RSO₃ ⁻ groups, where R issubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.

Target: An intended molecule to which a disclosed conformationallyrestricted cyanine fluorophore comprising a targeting agent is capableof specifically binding. Examples of targets include proteins andnucleic acid sequences present in tissue samples. A target area is anarea in which a target molecule is located or potentially located.

Targeting agent: An agent that promotes preferential or targeteddelivery to a target site, for example, a targeted location in asubject's body, such as a specific organ, organelle, physiologic system,tissue, or site of pathology such as a tumor, area of infection, or areaof tissue injury.

Targeting agents function by a variety of mechanisms, such as selectiveconcentration in a target site or by binding to a specific bindingpartner. Suitable targeting agents include, but are not limited to,proteins, polypeptides, peptides, glycoproteins and other glycoslyatedmolecules, oligonucleotides, phospholipids, lipoproteins, alkaloids, andsteroids. Exemplary targeting agents include antibodies, antibodyfragments, affibodies, aptamers, albumin, cytokines, lymphokines, growthfactors, hormones, enzymes, immune modulators, receptor proteins,antisense oligonucleotides, avidin, nano particles, and the like.Particularly useful of targeting agents are antibodies, nucleic acidsequences, and receptor ligands, although any pair of specific bindingpartners can be readily employed for this purpose.

TCEP: Tris(2-carboxyethyl)phosphine, a reducing agent.

TRABI: Temporal, radial-aperture-based intensity estimation.

Treat/treatment: As used herein, the terms “treat” and “treatment” meanto inhibit or reduce at least one sign or symptom associated with acondition, i.e., a disorder or disease. With respect to a tumor,treating may mean inhibiting tumor growth and/or reducing a tumorvolume. Treatment may, for example, produce a reduction in severity ofsome or all clinical symptoms of the tumor, a slower progression of thetumor (for example by prolonging the life of a subject having thetumor), a reduction in the number of tumor reoccurrence, an improvementin the overall health or well-being of the subject, or by otherparameters well known in the art that are specific to the particulardisorder or disease.

Trityl: A substituted or unsubstituted triphenyl methyl group, e.g.,Ph₃C—OR— or Ph₃CR— where R is aliphatic. Each phenyl group and Rindependently may be substituted or unsubstituted.

TSTU: N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate,a coupling agent.

II. Conformationally Restricted Cyanine Fluorophores

Embodiments of the disclosed conformationally restricted cyaninefluorophores have a chemical structure according to Formula I, or astereoisomer or pharmaceutically acceptable salt thereof:

wherein A is

and wherein each “*” designates an attachment point of A. The bondsrepresented by “

” are single or double bonds as needed to satisfy valence requirements.R¹-R⁹ and R¹¹ independently are H, deuterium, alkyl, heteroalkyl,—N(R^(a))₂, sulfonate, alkyl sulfonate, amino, aminoalkyl, trityl,—C(O)OR^(a), or a group comprising a conjugatable moiety, a targetingagent, or a drug, where each R^(a) independently is H, deuterium, alkylor heteroalkyl. R¹⁰ is H, deuterium, O, alkyl, aryl, amino, sulfonate,—C(O)OR^(b), —OR^(b), —N(R^(b))₂, heteroalkyl, heteroaryl, trityl, or agroup comprising a conjugatable moiety, a targeting agent, or a drug,where each R^(b) independently is H, deuterium, alkyl, heteroalkyl, arylor heteroaryl. Y¹ and Y² independently are C(R^(c))₂, N(R^(d)), S, O, orSe, wherein each R^(c) independently is H, deuterium, alkyl,—(OCH₂CH₂)_(x)OH where x is an integer ≥2, trityl, or a group comprisinga conjugatable moiety, a targeting agent, or a drug, and each R^(d)independently is H, deuterium, alkyl, or heteroalkyl. In some examples,the trityl group has a formula —(CH₂)₃₀C(Ph₂)(p-methoxyphenyl).

In any of the disclosed embodiments, an alkyl group or alkyl moiety ofan alkyl sulfonate or aminoalkyl group may be lower alkyl (C₁-C₁₀alkyl), C₁-C₅ alkyl, C₁-C₃ alkyl, methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅alkyl, C₆ alkyl, C₇ alkyl, C₈ alkyl, C₉ alkyl, C₁₀ alkyl. A heteroalkylgroup may have a chain length, including carbon atoms and heteroatoms,of from 1-10, 1-5, or 1-3, such as a chain length of 2, 3, 4, 5, 6, 7,8, 9, or 10.

In some embodiments, the conformationally restricted cyaninefluorophores have a chemical structure according to any one of FormulasIA-ID or a pharmaceutically acceptable salt thereof, wherein R¹-R¹¹, Y¹,and Y² are as previously described:

In certain embodiments, the conformationally restricted cyaninefluorophores have a chemical structure according to Formula II orFormula III, or a stereoisomer or pharmaceutically acceptable saltthereof, wherein R¹-R¹¹ and R^(c) are as previously described:

In any or all of the above embodiments, R¹-R⁹ and R¹¹ independently areH, deuterium, alkyl, heteroalkyl, —N(R^(a))₂, sulfonate, alkylsulfonate, amino, aminoalkyl, —C(O)OR^(a), or a group comprising aconjugatable moiety, a targeting agent, or a drug, where R^(a) is H,deuterium, alkyl or heteroalkyl. In some embodiments, at least one of R³and R⁶ is sulfonate, —C(O)OR^(a), trityl, or a group comprising aconjugatable moiety, a targeting agent, or a drug. In any or all of theforegoing embodiments, R¹, R², R⁴, R⁵, R⁷, and R⁸ may be H. In any orall of the foregoing embodiments, R⁹ may be H. In any or all of theforegoing embodiments, R¹ and R⁸, R² and R⁷, R³ and R⁶, R⁴ and R⁵,and/or R⁹ and R¹¹ may be the same. In certain embodiments, R¹, R², R⁴,R⁵, and R⁷-R¹¹ are H, and R³ and R⁶ independently are —SO₃ or —CO₂R^(a).In some examples, R³ and R⁶ are sulfonate.

In any or all of the above embodiments, R¹⁰ is H, deuterium, O, alkyl,aryl, amino, sulfonate, triflate, (—OS(O)₂CF₃), —C(O)OR^(b), —OR^(b),heteroalkyl, heteroaryl, trityl, or a group comprising a conjugatablemoiety, a targeting agent, or a drug, where R^(b) is H, deuterium,alkyl, heteroalkyl, aryl, or heteroaryl. In some embodiments, R¹⁰ is H,deuterium, O, alkyl, aryl, amino, sulfonate, triflate, (—OS(O)₂CF₃),—C(O)OR^(b), —OR^(b), heteroalkyl, heteroaryl, or a group comprising aconjugatable moiety, a targeting agent, or a drug, where R^(b) is H,deuterium, alkyl, heteroalkyl, aryl, or heteroaryl. In certainembodiments, R¹⁰ is H, O, aryl, —OR^(b), —N(R^(b))₂, or triflate. In anyor all of the foregoing embodiments, when the compound has a structureaccording to Formula II, R¹⁰ may be H. In any or all of the foregoingembodiments, when the compound has a structure according to Formula III,R¹¹ may be H.

In any or all of the above embodiments, Y¹ and Y² independently areC(R^(c))₂, N(R^(d)), S, O, or Se, wherein each R independently is H,deuterium, alkyl, —(OCH₂CH₂)_(x)OH where x is an integer ≥2, trityl, ora group comprising a conjugatable moiety, a targeting agent, or a drug,and each R^(d) independently is H, deuterium, alkyl, heteroalkyl, ortrityl. In some embodiments, Y¹ and Y² are C(R^(c))₂ and each R^(c)independently is C₁-C₃ alkyl, —(CH₂)_(n)C(O)R, or H, where n is aninteger ≥1 and R^(e) is a conjugatable moiety or a targeting agent. Inany or all of the foregoing embodiments, at least one R^(c) may be otherthan H. In any or all of the foregoing embodiments, each R^(c) may bethe same or each R^(d) may be the same. In one embodiment, Y¹ isC(R^(c))₂ where one R^(c) is a group comprising a conjugatable moiety, atargeting agent, or a drug and the other R^(c) is H, deuterium, oralkyl, and Y² is C(R^(c))₂ where each R independently is H or alkyl. Inan independent embodiment, Y¹ is C(R^(c))₂ where one R^(c) is a groupcomprising a conjugatable moiety, a targeting agent, or a drug and theother R^(c) is H or alkyl, and Y² is C(R^(c))₂ where one R^(c) is trityland the other R^(c) is H or alkyl. In another independent embodiment, Y²is C(R^(c))₂ where one R^(c) is a group comprising a conjugatablemoiety, a targeting agent, or a drug and the other R^(c) is H,deuterium, or alkyl, and Y¹ is C(R^(c))₂ where each R independently is Hor alkyl. In still another independent embodiment, Y² is C(R^(c))₂ whereone R^(c) is a group comprising a conjugatable moiety, a targetingagent, or a drug and the other R^(c) is H or alkyl, and Y¹ is C(R^(c))₂where one R^(c) is trityl and the other R^(c) is H or alkyl.

In any or all of the foregoing embodiments, where R^(c) is alkyl, thealkyl may be methyl.

In an independent embodiment, the compound has a chemical structureaccording to Formula II, R¹, R², R⁴, R⁵, R⁷, R⁸, and R^(c) are asdescribed above, R⁹ and R¹⁰ are H, and at least one of R³ and R⁶ is agroup comprising a conjugatable moiety, a targeting agent, or a drug. Inanother independent embodiment, the compound has a chemical structureaccording to Formula II, R¹, R², R⁴, R⁵, R⁷, and R⁸ are as describedabove, R⁹ and R¹⁰ are H, R³ and R⁶ are sulfonate, and at least one R^(c)is a group comprising a conjugatable moiety, a targeting agent, or adrug. In another independent embodiment, the compound has a chemicalstructure according to Formula III, R¹-R⁹ and R¹¹ are H, and R¹⁰ is H,O, triflate, aryl, —OR^(b), or —N(R^(b))₂. In yet another independentembodiment, the compound has a chemical structure according to FormulaIII, R¹-R⁹ and R¹¹ are H, and R¹⁰ is a group comprising a conjugatablemoiety, a targeting agent, or a drug. In another independent embodiment,the compound has a chemical structure according to Formula III, R¹, R²,R⁴, R⁵, R⁷, and R⁸ are as described above, R³ and R⁶ are sulfonate, R⁹and R¹¹ are H, R¹⁰ is H, O, triflate, aryl, —OR^(b), or —N(R^(b))₂, andat least one R^(c) is a group comprising a conjugatable moiety, atargeting agent, or a drug. Without wishing to be bound by a particulartheory of operation, in some embodiments, inclusion of sulfonate groups,e.g., at R³ and R⁶, may decrease aggregation of the compounds in aqueousmedia, exhibit improved antibody labeling properties, and/or exhibitimproved fluorescence emission compared to compounds without sulfonategroups.

In any or all of the above embodiments, a compound according to FormulaI, IA-ID, II, or III may include at least one group comprising aconjugatable moiety, a targeting agent, or a drug. Exemplary groupscomprising conjugatable moieties or targeting agents include, but arenot limited to, —(CH₂)_(n)C(O)R^(e), —(CH₂)_(n)N(H)R^(e),—(CH₂)_(n)N(H)C(O)R^(e), —(CH₂)_(n)C(O)N(H)R^(e), —(CH₂)_(n)C(O)SR^(e),—C(O)R^(e), —C(O)N(H)R^(e),—C(O)N(H)(CH₂CH₂O)_(m)(CH₂)_(n)C(O)R^(e)—N(H)C(O)R^(e), —N(H)R^(e), or—SR^(e) where m is an integer ≥1, n is an integer ≥1, and R^(e) is aconjugatable moiety or targeting agent.

Suitable conjugatable moieties include, but are not limited to,

where y is an integer ≥1, and phosphoramidites. In some examples, thephosphoramidite has a formula —(CH₂)₃OP(O(CH₂)CN)(N(i-Pr)₂) where i-Pris isopropyl. In certain embodiments, when the compound includes aphosphoramidite group on one half of the molecule, the compound may alsoinclude a trityl group on the other half of the molecule. For example,if Y¹ is substituted with a phosphoramidite group, then Y² may besubstituted with a trityl group.

Exemplary targeting agents include, but are not limited to, antibodies,ligands, peptides, nucleic acid strands, and the like. In some examples,the targeting agent is phalloidin, a bicyclic heptapeptide that binds toF-actin. In certain examples, the targeting agent is an antibody.Exemplary antibodies include antibodies capable of recognizing andbinding to a target molecule, such as a biomarker associated with adisease, infection, or environmental exposure. Biomarkers include, butare not limited to, proteins, peptides, lipids, metabolites, and nucleicacids. In some embodiments, the antibody is capable of recognizing andbinding to a tumor biomarker, such as a protein only found in or ontumor cells or to a cell-surface receptor associated with one or morecancers. For example, panitumumab is a human monoclonal antibody thatrecognizes and binds to human epidermal growth factor receptor 1 (HER1);HER1 is overexpressed in numerous tumor types and is also associatedwith some inflammatory diseases. Trastuzumab and pertuzumab aremonoclonal antibodies that bind to the HER2/neu receptor, which isover-expressed in some breast cancers. Brentuximab is a monoclonalantibody that targets a cell-membrane protein CD30, which is expressedin classical Hodgkin lymphoma and systemic anaplastic large celllymphoma.

Exemplary groups comprising a drug include, but are not limited to,groups having a formula -L₁-C(O)—X¹-drug, where L₁ is a linker moiety oris absent and X¹ is O, N(H), or N(CH₃). In one embodiment, L₁ is absent.In another embodiment, L₁ is O. In an independent embodiment, L₁ is arylor heteroaryl substituted with at least one substituent comprising asubstituted or unsubstituted aliphatic or heteroaliphatic moiety,wherein the aryl or heteroaryl ring is the site of attachment to theremainder of the conformationally restricted cyanine fluorophore and thesubstituent is bonded to the —C(O)—X¹-drug moiety. In some embodiments,the group comprising a drug is:

where X¹ is O, N(H), or N(CH₃), and R¹²-R¹⁹ independently are H, alkyl,—NO₂, —NR^(f) ₂, —NR^(f) ₃ ⁺, alkoxy, or sulfonate, wherein each R^(f)independently is H, halo, or alkyl. In certain embodiments, R¹²—R²³ areH. In some examples, the group comprising a drug is —C(O)—X¹-Drug. Thedrug can be any drug capable of conjugation to the remainder of thegroup. In some embodiments, the drug is a small-molecule drug, e.g., adrug having a molecular weight <1,000 Daltons. In certain embodiments,the drug moiety is an anti-cancer drug.

Nonlimiting examples of compounds according to Formula I include:

Embodiments of the disclosed conformationally restricted cyaninefluorophores exhibit improved quantum yields and/or extendedfluorescence lifetimes relative to corresponding unrestrainedpentamethine and heptamethine cyanines. In some embodiments, the quantumyield and/or fluorescence lifetime is at least 1.1×, at least 1.5×, atleast 2×, at least 3×, at least 4×, or at least 5× greater than thequantum yield and/or fluorescence lifetime of the correspondingnon-restricted cyanine fluorophore. The quantum yield and/orfluorescence lifetime may be 1.1-10×, such as 1.5-8×, 1.5-5×, or 2-5×greater than the quantum yield and/or fluorescence lifetime of thecorresponding non-restricted cyanine fluorophore. Advantageously, themaximum wavelengths for absorption and emission may be red-shiftedcompared to the corresponding non-restricted cyanine fluorophore. Incertain embodiments, λ_(max) and/or λ_(em) are red-shifted by at least10 nm, at least 20 nm, or at least 30 nm, such as from 10-50 nm, 10-40nm, 10-30 nm, or 10-20 nm relative to the λ_(max) and/or λ_(em) valuesof the corresponding non-restricted cyanine fluorophore. Additionally,some embodiments of the disclosed conformationally restricted cyaninefluorophores recover from hydride reduction with superior efficiencyrelative to existing far-red and/or near-IR cyanines. In certainembodiments, these properties enable PALM-like SMLM, providing excellentphoton counts without recourse to high thiol, deoxygenated buffer.

III. Pharmaceutical Compositions

This disclosure also includes pharmaceutical compositions comprising atleast one conformationally restricted cyanine fluorophore as disclosedherein. Some embodiments of the pharmaceutical compositions include apharmaceutically acceptable carrier and at least one conformationallyrestricted cyanine fluorophore. Useful pharmaceutically acceptablecarriers and excipients are known in the art.

The pharmaceutical compositions comprising one or more conformationallyrestricted cyanine fluorophores may be formulated in a variety of waysdepending, for example, on the mode of administration and/or on thelocation to be imaged. Parenteral formulations may comprise injectablefluids that are pharmaceutically and physiologically acceptable fluidvehicles such as water, physiological saline, other balanced saltsolutions, aqueous dextrose, glycerol or the like. Excipients mayinclude, for example, nonionic solubilizers, such as Cremophor®, orproteins, such as human serum albumin or plasma preparations. Ifdesired, the pharmaceutical composition to be administered may alsocontain non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, forexample, sodium acetate or sorbitan monolaurate.

The form of the pharmaceutical composition will be determined by themode of administration chosen. Embodiments of the disclosedpharmaceutical compositions may take a form suitable for virtually anymode of administration, including, for example, topical, ocular, oral,buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc.,or a form suitable for administration by inhalation or insufflation.Generally, embodiments of the disclosed pharmaceutical compositions willbe administered by injection, systemically, or orally.

Useful injectable preparations include sterile suspensions, solutions oremulsions of the active compound(s) in aqueous or oily vehicles. Thecompositions may also contain formulating agents, such as suspending,stabilizing and/or dispersing agent. The formulations for injection maybe presented in unit dosage form, e.g., in ampules or in multidosecontainers, and may contain added preservatives. The composition maytake such forms as suspension, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. For example, parenteraladministration may be done by bolus injection or continuous infusion.Alternatively, the conformationally restricted cyanine fluorophore maybe in powder form for reconstitution with a suitable vehicle, e.g.sterile water, before use.

Systemic formulations include those designed for administration byinjection, e.g., subcutaneous, intravenous, intramuscular, intrathecalor intraperitoneal injection, as well as those designed for transdermal,transmucosal, oral or pulmonary administration.

Oral formulations may be liquid (e.g., syrups, solutions orsuspensions), or solid (e.g., powder, tablets, or capsules). Oralformulations may be coupled with targeting ligands for crossing theendothelial barrier. Some conformationally restricted cyaninefluorophore formulations may be dried, e.g., by spray-drying with adisaccharide, to form conformationally restricted cyanine fluorophorepowders. Solid compositions prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, mannitol, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets may be coated by methods well known in the art with, forexample, sugars, films or enteric coatings. Actual methods of preparingsuch dosage forms are known, or will be apparent, to those skilled inthe art.

Liquid preparations for oral administration may take the form of, forexample, elixirs, solutions, syrups or suspensions. Such liquidpreparations may be prepared by conventional means with pharmaceuticallyacceptable additives such as suspending agents (e.g., sorbitol syrup,cellulose derivatives or hydrogenated edible fats); emulsifying agents(e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, ethyl alcohol, Cremophor® or fractionated vegetable oils); andpreservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbicacid). The preparations may also contain buffer salts, preservatives,flavoring, coloring and sweetening agents as appropriate. Preparationsfor oral administration may be suitably formulated to give controlledrelease of the fluorophore, as is well known.

For rectal and vaginal routes of administration, the conformationallyrestricted cyanine fluorophore(s) may be formulated as solutions (forretention enemas) suppositories or ointments containing conventionalsuppository bases such as cocoa butter or other glycerides.

For nasal administration or administration by inhalation orinsufflation, the conformationally restricted cyanine fluorophore(s) canbe conveniently delivered in the form of an aerosol spray or mist frompressurized packs or a nebulizer with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or othersuitable gas. In the case of a pressurized aerosol, the dosage unit maybe determined by providing a valve to deliver a metered amount.

Certain embodiments of the pharmaceutical compositions comprisingconformationally restricted cyanine fluorophores as described herein maybe formulated in unit dosage form suitable for individual administrationof precise dosages. The pharmaceutical compositions may, if desired, bepresented in a pack or dispenser device which may contain one or moreunit dosage forms containing the conformationally restricted cyaninefluorophore. The pack may, for example, comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration.

The amount of conformationally restricted cyanine fluorophoreadministered will depend at least in part on the subject being treated,the target (e.g., the size, location, and characteristics of a tumor),and the manner of administration, and may be determined as is known tothose skilled in the art of pharmaceutical composition and/or contrastagent administration. Within these bounds, the formulation to beadministered will contain a quantity of the conformationally restrictedcyanine fluorophore disclosed herein in an amount effective to enablevisualization of the conformationally restricted cyanine fluorophore bysuitable means after administration to the subject. In certainembodiments, the conformationally restricted cyanine fluorophorecomprises a drug bound to the molecule, and the formulation to beadministered will contain a quantity of the drug bound to theconformationally restricted cyanine fluorophore effective to provide atherapeutically effective dose of the drug to the subject being treated.

In some embodiments, the pharmaceutical composition includes a secondagent other than the conformationally restricted cyanine fluorophore.The second agent may be, for example, an anti-tumor agent or anangiogenesis inhibitor.

IV. Synthesis

Conformationally restricted trimethine cyanine dyes have beensynthesized previously by treating a trimethine cyanine dye in mild acidsolution (e.g., acetic acid) under refluxing conditions or in a strongermineral acid solution under milder conditions whereupon a “rigidized”carbocyanine dye precipitates from solution (see, e.g., WO99/31181).This synthetic strategy, however, cannot be extended to far-red andnear-IR cyanines such as pentamethine and heptamethine cyanines.Heretofore, no synthetic strategy for making conformationally restrictedcyanine fluorophores according to Formula I was known.

Disclosed herein are embodiments of methods for making conformationallyrestricted pentamethine and heptamethine cyanine fluorophores.Embodiments of a method for making conformationally restrictedpentamethine cyanine fluorophores, i.e., compounds according to FormulaI wherein A is

proceed via a cyclization cascade of a protected dialdehyde precursor,which is accessed through chemoselective olefin metathesis. FIG. 1illustrates one embodiment of a retrosynthetic pathway for making aconformationally restricted pentamethine cyanine fluorophore. In the keyreaction, a protected dialdehyde undergoes intramolecular Michaeladdition followed by a dihydropyran ring-forming cascade. As the cyaninepolyene is incompatible with nucleophilic olefination methods, acritical challenge is the chemoselective introduction of the sensitiveα,β-unsaturated aldehyde motif (or synthetic equivalent). Across-metathesis reaction was ultimately found to provide a suitablemethod to install an unsaturated acetal.

FIG. 2 shows an exemplary synthetic scheme for preparing one embodimentof an unsubstituted, conformationally restricted pentamethine cyaninefluorophore. Precursor 1 is prepared from N-alkylated indolenines asdescribed in more detail below. Cross-metathesis using Grubbs secondgeneration catalyst and acrolein dimethyl acetal in dichloromethaneprovided compound 3 following purification. Compound 3 undergoestetracyclization in a heated (e.g., 70° C.) acidified chloroformsolution to provide compound 4 as a single diastereomer. Alternatively,reaction of compound 3 in a cold (e.g., −78° C.) solution of borontribromide in dichloromethane provides compound 4 and another compound,which can be converted to compound 4 in a heated (e.g., 60° C.) solutionof 1:3 methanol:0.3 M HCl.

FIG. 3 shows an exemplary synthetic scheme for making a conjugatablevariant of a conformationally restricted pentamethine cyaninefluorophore. Cross-metathesis using Hoyveda-Grubbs second generationcatalyst proceeds efficiently between compound 5 and compound 2 at roomtemperature to provide compound 6, which can be used without extensivepurification. Tetracyclization of compound 6 proceeds in a cold (e.g.,−78° C.) solution of boron tribromide in dichloromethane to provide amixture including compound 7. Equilibration in a heated (e.g., 60° C.)solution of 1:3 methanol:0.3 M HCl provides a methyl ester 6, whichafter saponification and purification provides compound 7. To prepare acompound suitable for bioconjugation, compound 7 may be converted to acarboxylic acid 8 through an amide coupling sequence. In the example ofFIG. 3, phalloidin was conjugated to the molecule byN-hydroxysuccinimide-ester generation and amide bond formation toprovide a phalloidin conjugate 9.

In some embodiments, the method includes combining a solution comprisinga compound according to Formula IV with 3-buten-1-yltrifluoromethanesulfonate to produce a compound according to Formula V

A solution comprising the compound according to Formula V and a compoundaccording to Formula VI is combined withN-((1E,3Z)-3-(phenylamino)propo-1-en-1-yl)aniline orN-((1E,3E)-3-(phenylimino)prop-1-en-1-yl)aniline (e.g., malonaldehydebis(phenylimine) monohydrochloride) to form a compound according toFormula VII

A solution comprising the compound according to Formula VII is combinedwith 3,3-dimethoxy-1-propene in the presence of a ruthenium catalyst(e.g., Grubbs second generationcatalyst-1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene)-dichloro(phenylmethyleneXtricyclohexylphosphine)ruthenium,or Hoyveda-Grubbs second generationcatalyst-1,3-bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene)-dichloro(o-isopropoxyphenylmethylene)ruthenium)to provide a compound according to Formula VIII

The compound according to Formula VIII is combined with (i) a mixture ofCHCl₃ and H₂SO₄ or (ii) BBr₃ in CH₂Cl₂ to provide a compound accordingto Formula IX:

Embodiments of a method for making conformationally restrictedheptamethine cyanine fluorophores, i.e., compounds according to FormulaI wherein A is

include combining a solution comprising a compound according to FormulaIV with 3-buten-1-yl trifluoromethanesulfonate to produce a compoundaccording to Formula V, and combining a solution comprising a compoundaccording to Formula X with 3-buten-1-yl trifluoromethanesulfonate toproduce a compound according to Formula XI:

In some embodiments, it may be desirable to produce a symmetricalconformationally restricted cyanine fluorophore. In such embodiments,the compounds according to Formula IV and X are the same, therebyproducing compounds according to Formulas V and XI that are the same. Inthese embodiments, a single step of combining a solution comprising acompound according to Formula IV/X with 3-buten-1-yltrifluoromethanesulfonate to produce a compound according to FormulaV/XI may be performed.

A solution comprising the compound according to Formula V and thecompound according to Formula XI (if different than the compoundaccording to Formula V) is combined with(1E,4E)-1,5-bis(dimethylamino)-penta-1,4-dien-3-one to produce acompound according to Formula XII:

A solution comprising the compound according to Formula XII is combinedwith 3,3-dimethoxy-1-propene in the presence of a ruthenium catalyst(e.g., Grubbs second generationcatalyst-1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene)-dichloro(phenylmethyleneXtricyclohexylphosphine)ruthenium,or Hoyveda-Grubbs second generationcatalyst-1,3-bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene)-dichloro(o-isopropoxyphenylmethylene)ruthenium)to provide a compound according to Formula XIII:

A solution comprising the compound according to Formula XIII is combinedwith an acidic tetrahydrofuran solution (e.g., 1 N HCl intetrahydrofuran) to provide a compound according to Formula XIV:

The ketone group of Formula XIV can be modified as desired to providevariations in the structure. Several variations can be made following aninitial conversion of the ketone group to triflate. The triflate groupis introduced by combining a solution comprising the compound accordingto Formula XIV with trifluoromethanesulfonic anhydride (Tf₂O) to providea compound according to Formula XV:

In one embodiment, the triflate group is replaced with an aryl group bycombining a solution comprising the compound according to Formula XVwith R^(g)—C₆H₄—B(OH)₂ in the presence of a palladium catalyst toprovide a compound according to Formula XVI:

where R^(g) is R^(a), —COOR^(a), or —OR^(a), and R^(a) is H, deuterium,alkyl or heteroalkyl.

In another embodiment, the triflate group is replaced with an aminogroup by combining a solution comprising the compound according toFormula XV with an amine having a formula NH(R²⁰)(R²¹) to provide acompound according to formula XVII:

where R²⁰ and R²¹ independently are H, deuterium, alkyl, heteroalkyl,aryl or heteroaryl. This process can be performed in the presence orabsence of a palladium catalyst.

In some examples, R²⁰ is —(CR^(h) ₂)_(n)-CH₂OH where each R^(h)independently is H, deuterium, halo, alkyl, or aryl, and n is 1, 2, 3,or 4; and R²¹ is H, deuterium, alkyl, heteroalkyl, aryl, or heteroaryl.Such compounds have a structure according to Formula XVIIa:

In certain embodiments, each R^(h) is H and/or R²¹ is H. In any or allof the foregoing embodiments, n may be 1 or 2.

In some embodiments, a compound according to Formula XVIIa can undergoan N- to O-rearrangement when reacted with an electrophile under basicconditions. Thus, in certain examples, a solution comprising a compoundaccording to Formula XVIIa is combined with a compound comprising anelectrophilic group R²² under basic conditions to provide a compoundaccording to Formula XVIII:

Suitable electrophilic groups R²² include, but are not limited to, amaleimidyl-containing group, a succinimidyl-containing group, optionallysubstituted alkoxy, optionally substituted alkyl carbonyl, optionallysubstituted alkoxy carbonyl, a biomolecule-containing group, or acombination thereof. Exemplary maleimidyl- and succinimidyl-containinggroups may further include a carbonyl, alkyl carbonyl, alkoxy, or alkoxycarbonyl group attached to the ring nitrogen. In some embodiments, R²²is formed by a combination of two groups that participate in therearrangement, e.g., glutaric anhydride and N-hydroxysuccinimide maycombine to form the succinimidyl-containing group—(O)C(CH₂)₃C(O)O—NC₄H₄O₂.

Exemplary solvents for the reaction include, but are not limited to,tetrahydrofuran (THF), N,N-dimethylformamide (DMF), dichloromethane(DCM), water, and combinations thereof.

Suitable bases include inorganic and organic bases. Exemplary basesinclude, but are not limited to, carbonates (e.g., K₂CO₃, Na₂CO₃),hydrogen carbonates (e.g., KHCO₃, NaHCO₃), hydroxides (e.g., KOH, NaOH),and organic amines (e.g., 4-dimethylaminopyridine (DMAP),N,N-diisopropylethylamine (DIPEA),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, EDAC, or EDCI), andN-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminiumhexafluorophosphate N-oxide (HATU)).

Exemplary compounds comprising R²² include, but are not limited tochloroformates, cyclic anhydrides, alkyl halides, carboxylic acids, andtetrafluoroborates. In some embodiments, the carboxylic acids areactivated carboxylic acids, such as carboxylic acids preactivated withHATU and DIPEA before addition to the solution comprising the compoundaccording to Formula XVIIa.

Effective conditions may include reacting at a reaction temperatureranging from room temperature (20-26° C.) to 100° C. for a time rangingfrom a few minutes to several hours. In certain examples, the reactiontemperature ranges from room temperature to 90° C., and the time rangesfrom 10 minutes to 18 hours. In one example, the solution is irradiatedwith microwave irradiation at the reaction temperature. In variousembodiments, the solution is stirred gently, stirred vigorously, or notstirred. The reaction may proceed in a sealed vessel under an inertatmosphere (e.g., argon, nitrogen). Completion of the reaction may bemonitored by any suitable means including, but not limited to, a visualcolor change or LC/MS. The compound according to Formula XVIII isrecovered, and optionally purified, by suitable means. In someembodiments, the compound is recovered and/or purified by extraction,precipitation, evaporation, ion exchange, chromatography (e.g., silicagel chromatography, HPLC), and combinations thereof.

Some embodiments of the compounds disclosed herein are suitable forfurther conjugation to a targeting agent, such as a biomolecule. Forexample, when R²² terminates in a succinimidyl moiety, a maleimidylmoiety, —COOH, or —COO⁻, a biomolecule may be conjugated to theconformationally restricted cyanine fluorophore by methods known to aperson of ordinary skill in the art, e.g., via a bioconjugation reactionmediated by N,N′-disuccinimidyl carbonate. Suitable biomoleculesinclude, but are not limited to, antibodies, peptides, amino acids,proteins, and haptens.

V. Methods of Use

Embodiments of the disclosed compounds according to Formula I may beuseful for live-cell localization and tracking applications.Additionally, some embodiments of the disclosed compounds may be usefulfor sensing reactive oxygen species (ROS). In certain embodiments,inclusion of sulfonate or other polar functional groups may facilitateantibody labeling.

Furthermore, some embodiments of the disclosed compounds may becell-permeable, an advantage for live-cell studies. Investigative,diagnostic, and theranostic uses are within the scope of the disclosure.

Compounds according to Formula I may be utilized in in vitro, ex vivo,and in vivo localization and tracking applications. In some embodiments,a compound according to Formula I that comprises at least one targetingagent at any of R¹-R¹¹, Y¹, or Y² is combined with a sample comprising atarget capable of binding with the targeting agent, and the target isimaged by visualizing the compound. The sample may be, for instance, atissue sample, a biological fluid (e.g., blood, urine, saliva), or atarget area within a subject such as a location of a known or suspectedtumor. Advantageously, combining the compound with the sample isperformed under conditions (temperature, pH, concentration, etc.)effective to provide binding of the targeting agent and target. After aperiod time effective for binding to occur, which may range from a fewseconds to several days, the compound is visualized. Prior tovisualization, excess, unbound compound may be removed from the sample,e.g., by washing the sample under conditions effective to remove unboundcompound without disrupting compound molecules bound to the target or bywaiting a sufficient period of time (such as a few hours or days) forunbound compound to be eliminated from the target area in vivo. Incertain embodiments, the compound according to Formula I is reducedprior to visualization. Suitable reducing agents include, but are notlimited to, hydrides such as sodium borohydride (NaBH₄). The reductionoccurs in moiety A as shown below.

-   -   reduced A:

In some embodiments, visualization comprises irradiating the sample or atargeted portion of a subject with targeted application of a quantity oflight having a wavelength in the visible, far-red, or near-infraredrange and a selected intensity, wherein the quantity of light issufficient to produce fluorescence of the compound, and detecting anyfluorescence emitted by the compound. Advantageously, the light has awavelength at or near a maximum absorption wavelength of the compoundaccording to Formula I. For example, the sample may be irradiated withlight having a wavelength within a range of 600 nm to 2500 nm, such asfrom 600-900 nm, or 600-700 nm. In some embodiments, the light source isa laser. Suitable light intensities may range from 1 mW/cm² to 1000mW/cm², such as 1-750 mW/cm² or 300-700 mW/cm², depending on the targetsite and method of application. Near-infrared light sources can beobtained from commercial sources, including Thorlabs (Newton, N.J.),Laser Components, USA (Hudson, N.H.), ProPhotonix (Salem, N.H.) andothers. In some embodiments, the effective quantity of far-red or NIRlight is 10-250 J, such as 10-200 J, 10-150 J, or 10-100 J.

In some embodiments, visualization may include techniques such asfluoroscopy, single-molecule localization microscopy (SMLM),photo-activated localization microscopy (PALM), stochastic opticalreconstruction microscopy (STORM), direct stochastic opticalreconstruction microscopy (dSTORM), biplane imaging (BP), temporalradial-aperture based intensity estimation (TRABI), fluorescenceresonance energy transfer (FRET), and combinations thereof.

In some embodiments, an effective amount of a compound according toFormula I or a pharmaceutical composition comprising the compound isadministered to a subject suspected of having a condition that may bedetected and/or evaluated by visualizing a fluorophore bound to a target(e.g., a tumor) within the subject. Advantageously, the compoundaccording to Formula I may include a targeting agent capable of bindingto the target within the subject. Administration is performed by anysuitable method, e.g., intravenous, intra-arterial, intramuscular,intratumoral, or subcutaneous injection, or oral, intranasal, orsublingual administration. The administered compound is subsequentlyirradiated by targeted application of a quantity of light having awavelength in the far-red or near-infrared range and a selectedintensity to a target area of the subject, wherein the quantity of lightis sufficient to excite the compound according to Formula I. Whenirradiating a target area (e.g., an area proximate a tumor), theeffective quantity of far-red or NIR light may be 1-250 J/cm², such as1-250 J/cm², such as 5-250 J/cm², 10-250 J/cm², 10-200 J/cm², 10-150J/cm², 10-100 J/cm², or 30-100 J/cm². Any fluorescence from the compoundin the targeted portion of the subject is detected, thereby diagnosingthe subject as having the condition.

In certain theranostic embodiments, the condition is a tumor and thetargeted portion of the subject includes the tumor site. Theadministered compound is visualized by exposing the tumor to lighthaving a wavelength and intensity sufficient to induce fluorescence ofthe compound. Irradiation may be performed by external application oflight to a targeted area of a subject. Far-red or NIR light is capableof penetrating transcutaneously into tissue to a depth of severalcentimeters. In other embodiments, irradiation may be performed byinternal application of light, such as by using an endoscope, a fiberoptic catheter, or an implantable fluorescence device. Internalapplication may be used when the target tissue, such as a tumor, islocated at a depth that is unsuitable for external light application.For example, an endoscope may be used for light delivery into the lungs,stomach, or bladder. In some examples, the tumor site is exposed bysurgical incision prior to exposing the tumor to light. The tumor may beexcised using the area of fluorescence as guidance. In one embodiment,at least a portion of the tumor is excised from the subject beforeadministering the therapeutically effective amount of the compound orthe pharmaceutical composition comprising the compound to the subject.In an independent embodiment, the therapeutically effective amount ofthe compound or the pharmaceutical composition comprising the compoundis administered to the subject before surgical excision of the tumor ora portion thereof.

The surface area for light application is generally selected to includetarget tissue, e.g., a tumor or portion of a tumor, or an area of skinexternal to the target tissue. When targeted application of externallight is desired for an in vivo biological sample, the surface area canbe controlled by use of an appropriate light applicator, such as amicro-lens, a Fresnel lens, or a diffuser arrangement. For targetedinternal light application, a desired endoscope or fiber optic catheterdiameter can be selected. In some applications, an indwelling catheterfilled with a light scattering solution may be internally placedproximate the target tissue, and an optical fiber light source may beinserted into the catheter (see, e.g., Madsen et al., Lasers in Surgeryand Medicine 2001, 29, 406-412).

In another embodiment, an in vitro or ex vivo evaluation may beperformed to determine whether a compound according to Formula I willeffectively bind to a tissue sample obtained from a subject having, orsuspected of having, a condition that may be visualized by the compound.The compound comprises a targeting agent thought to be capable ofbinding to or associating with a target molecule indicative of orassociated with the condition. In one non-limiting example, thetargeting agent is a receptor ligand or antibody capable of binding to atarget receptor. The compound is combined with the tissue sample, andthe sample is subsequently irradiated with an effective amount ofnear-IR light. In one embodiment, the tissue sample is washed to removeexcess, unbound compound, and fluorescence of the tissue sample isassessed. Fluorescence indicates that the compound has bound to thetissue sample.

In certain embodiments, a compound according to Formula I may beutilized to visualize shapes and/or structures within a cell. A compoundaccording to Formula I may include a targeting agent capable of bindingto a desired component within a cell, e.g., a fixed or permeabilizedcell. For example, the compound may include phalloidin as a targetingagent that binds to F-actin. Visualization of F-actin is useful forshowing the overall shape and structure of a cell.

In an independent embodiment, a compound according to Formula I may beutilized to detect and/or measure the presence of reactive oxygenspecies (ROS) including superoxide and hydroxide radicals, in a sample.ROS have been implicated in a variety of inflammatory diseases, such ascancer and atherosclerosis. ROS detection can be performed in vitro, exvivo, or in vivo. In certain embodiments, the compound may be reducedprior to contacting a sample with the compound. ROS present in thesample may oxidize the compound to recreate the fluorophore, which isdetected by any suitable method. Fluorescence indicates the presence ofROS in the sample. The intensity of the fluorescence may be correlatedto the concentration of ROS in the sample.

VI. Examples General Materials and Methods

All commercially obtained reagents were used as received.2,3,3-trimethyl indoline (S1), malonaldehyde bis(phenylimine)monohydrochloride, Grubb's 2^(nd) generation and Hoyveda-Grubbs 2^(nd)generation catalysts were purchased from Sigma-Aldrich (St. Louis, Mo.).Acrolein dimethyl acetal (2) was purchased from TCI America (Portland,Oreg.). 4-hydrazinylbenzoic acid (S5) was ordered from Oakwood Chemical(Estill, S.C.). 2-(2-bromoethyl)-1,3-dioxolane was purchased from AcrosOrganics (Geel, Belgium). NH₂—PEGs-COOH was purchased from ThermoScientific (Waltham, Mass.). Aminophalloidin tosylate was purchased fromEnzo, Inc. (Farmingdale, N.Y.). Compounds S2, S4, and S8 weresynthesized according to known procedures (Hanessian et al., J. Am.Chem. Soc. 2004, 126 (19), 6064-71; Hall et al., Nucleic Acids Res.2012, 40 (14), e108; Park et al., Bioconjugate Chem. 2012, 23 (3),350-362). Normal phase (60 Å, 20-40 μm, RediSep® Rf Gold® silica or 60Å, 35-70 μm, RediSep® Rf silica) and reversed phase (100 Å, 20-40 micronparticle size, RediSep® Rf Gold® Reversed-phase C18 or C18Aq) flashcolumn chromatography was performed on a CombiFlash® Rf 200i (TeledyneIsco, Inc., Lincoln, Nebr.). Reversed-phase preparative HPLC wasperformed using an Agilent 1260 Infinity II LC system utilizing aSunFire Prep C18 column (100 Å, 5 μm, 10×150 mm) obtained from Waters,Co (Milford, Mass.). High-resolution LC/MS analyses were conducted on aThermo-Fisher LTQ-Orbitrap-XL hybrid mass spectrometer system with anIon MAX API electrospray ion source in negative ion mode. AnalyticalLC/MS was performed using a Shimadzu LCMS-2020 Single Quadrupoleutilizing a Kinetex C18 column (100 Å, 2.6 μm, 2.1×50 mm) obtained fromPhenomenex, Inc (Torrance, Calif.). Runs employed a gradient of 0-90%MeCN/0.1% aqueous formic acid over 4.5 min at a flow rate of 0.2 mL/min.¹H NMR and ¹³C NMR spectra were recorded on Bruker spectrometers (at 400or 500 MHz or at 100 or 125 MHz) and are reported relative to deuteratedsolvent signals. Data for ¹H NMR spectra are reported as follows:chemical shift (6 ppm), multiplicity, coupling constant (Hz), andintegration. Data for ¹³C NMR spectra are reported in terms of chemicalshift. Absorbance curves were obtained on a Shimadzu UV-2550spectrophotometer operated by UVProbe 2.32 software. Molar absorptioncoefficients (e) were determined in PBS (50 mM, pH 7.4) or methanol(MeOH) using Beer's law, from plots of absorbance vs. concentration.Measurements were performed in 10 mm path length quartz cuvettes (Hellma111-QS) that were maintained at room temperature, and reported valuesare averages (n ≥3). Fluorescence traces were recorded on a PTIQuantaMaster steady-state spectrofluorimeter operated by FelixGX 4.2.2software, with 4 nm excitation and emission slit widths, and a 0.1 sintegration rate. A Quantaurus-QY spectrometer (Hamamatsu, model C11374)was used to determine absolute fluorescence quantum yields (Φ_(F))(Suzuki et al., Phys. Chem. Chem. Phys. 11 2009, 9850-9860). Thisinstrument uses an integrating sphere to measure photons absorbed andemitted by a sample.

Measurements were carried out at a concentration of 250 nM in eitherMeOH or PBS (50 mM, pH 7.4) and self-absorption corrections wereperformed using the instrument software. Reported values are averages(n≥5). Data analysis and curve fitting were performed using MS Excel2011 and GraphPad Prism 7. Light intensity measurements were performedwith a Thorlabs PM200 optical power and energy meter fitted with anS120VC standard Si photodiode power sensor (200-1100 nm, 50 nW-50 mW).See JOC Standard Abbreviations and Acronyms for abbreviations(http://pubs.acs.org/paragonplus/submission/joceah/-joceah_abbreviations.pdf).

Single-molecule imaging was performed on a custom-built objective-typeTIRF setup as described elsewhere (van de Linde et al., Nature Protocols2011, 6:991), employing highly inclined illumination. An invertedmicroscope (IX71, Olympus) equipped with a nosepiece stage (IX2-NPS,Olympus) and a 60× oil objective (NA 1.45 PlanApo, Olympus) was used.TRABI-Biplane imaging was performed as described elsewhere (Franke etal., Nature Methods 2017 14:41). In brief, a two-channel image splitterwith twofold magnification (TwinCam, Cairn Research) equipped with a50/50 beamsplitter (Cairn Research) and two EMCCD cameras (Ixon 897 andIxon Ultra 897, Andor), whose focal planes were separated by 300 nm wereused. Cameras were synchronized by a pulse generator (DG535, StanfordResearch Systems). 3D calibration experiments were performed by movingthe objective with a piezo scanner (Pifoc, Physik Instrumente) drivenwith a LVPZT servo controller (E-662, Physik Instrumente). Z-coordinatesfrom TRABI-Biplane imaging were corrected for the refractive indexmismatch by a scaling factor of 0.71 (refractive index of buffern_(b)=1.34 and substrate (glass) n_(s)=1.52, numerical aperture ofNA=1.45).

Example 1 Exemplary Syntheses for Conformationally RestrictedPentamethine Cyanine Fluorophores

(S3): To a solution of 2,3,3-trimethyl indoline (S1, 2.0 g, 12.5 mmol,1.0 eq) in CH₂Cl₂ (50 mL, 0.25M) at 0° C. was added S2 (3.0 g, 15 mmol,1.2 eq). The solution was allowed to warm to room temperature and standfor 1 hour. The reaction volume was reduced to approximately 10 mL, andthen purified by normal phase chromatography (24 g silica column, 0-15%MeOH/CH₂Cl₂) to afford S3 (2.5 g, 6.9 mmol, 55%) as a dark purple solid.¹H NMR (400 MHz, CDCl₃) δ 7.71-7.65 (m, 1H), 7.62-7.54 (m, 3H), 5.82(ddt, J=17.3, 10.1, 7.3 Hz, 1H), 5.07 (ddd, J=10.1, 1.5, 0.9 Hz, 1H),4.90 (dq, J=17.0, 1.4 Hz, 1H), 4.65 (t, J=6.7 Hz, 2H), 2.82 (s, 3H),2.74 (q, J=6.9 Hz, 2H), 1.57 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 196.7,141.6, 140.7, 132.2, 130.1, 129.5, 123.2, 120.3, 115.4, 54.6, 47.6,32.2, 23.2, 14.9. HRMS (ESI) calculated for C₁₅H₂₀N (M⁺) 214.1590,observed 214.1588.

(1): To a solution of S3 (1.0 g, 5.0 mmol, 1.0 eq), S4 (1.3 g, 5.0 mmol,1.0 eq), malonaldehyde bis (phenylimine) monohydrochloride (1.3 g, 5.0mmol, 1.0 eq), and Et₃N (3.6 mL, mmol, 5 eq) in CH₂Cl₂ (50 mL) was addedAc₂O (1.4 mL, 15 mmol, 3 eq). The solution was allowed to stand for 2hours at room temperature. To the dark blue solution was then addedaqueous NaI (50 mL, 0.4 M). The mixture was stirred vigorously for 18hours and separated with CH₂Cl₂ (2×100 mL), dried over NaHSO₄ andconcentrated under reduced pressure. The resulting blue residue waspurified by normal phase column chromatography (40 g silica column,25-75% ethyl acetate/CH₂Cl₂) to provide 1 (2^(nd) eluting peak) as ablue solid (1.20 g, 1.9 mmol, 38%). ¹H NMR (400 MHz, CD₃OD) δ 8.27 (td,J=13.1, 3.0 Hz, 2H), 7.51 (d, J=7.4 Hz, 2H), 7.43 (t, J=7.7 Hz, 2H),7.32 (d, J=8.0 Hz, 2H), 7.28 (t, J=7.5 Hz, 2H), 6.65 (t, J=12.4 Hz, 1H),6.34 (dd, J=13.7, 6.1 Hz, 2H), 5.92 (ddt, J=17.2, 10.1, 7.1 Hz, 1H),5.14-5.03 (m, 2H), 5.01 (t, J=3.9 Hz, 1H), 4.25 (dt, J=14.6, 7.0 Hz,4H), 4.02-3.81 (m, 4H), 2.62 (q, J=7.0 Hz, 2H), 2.21 (td, J=7.1, 3.9 Hz,2H), 1.74 (s, 12H). ¹³C NMR (101 MHz, CD₃OD) δ 173.6, 173.2, 154.1,154.0, 142.1, 142.1, 141.2, 141.2, 133.7, 128.3, 128.2, 125.2, 124.8,124.8, 120.0, 122.0, 117.4, 110.9, 110.6, 103.5, 103.1, 101.6, 64.7,49.2, 49.2, 42.7, 38.6, 31.6, 30.2, 26.7, 26.3. HRMS (ESI) calculatedfor C₃₄H₄₁N₂O₂ (M⁺) 509.3163, observed 509.3157.

(3): To a dark blue solution of 1 (80 mg, 0.13 mmol, 1.0 eq) and Grubb's2^(nd) generation catalyst (33 mg, 0.039 mmol, 0.3 eq) in CH₂Cl₂ (16 mL,0.01 M) was added acrolein dimethyl acetal (2, 149 μL, 1.3 mmol, 10 eq)under argon after under degassing with vacuum. This reaction wasrefluxed at 40° C. for 5.5 hours under argon and static vacuum, whichwas reapplied approximately once per hour. After cooling to roomtemperature, saturated aqueous NaCl (20 mL) was added and stirredvigorously for 18 hours. The biphasic mixture was then separated,extracted with CH₂Cl₂ (3×20 mL), dried over Na₂SO₄, and evaporated underreduced pressure. The dark blue residue was purified by normal phasechromatography (12 g silica column, 0-20% MeOH/CH₂Cl₂) to provide 3(2^(nd) eluting peak, 37 mg, 0.060 mmol, 48%). The first eluting peak,which contained impure product, was combined and again stirred overnightwith saturated aqueous NaCl (20 mL). The organic extraction in CH₂Cl₂and normal phase purification was repeated to yield 3 (11 mg, 0.018mmol, 14%). The purified product from both columns was combined toafford 3 (48 mg, 0.078 mmol, 62%) as a blue solid. ¹H NMR (500 MHz,CD₃OD) δ 8.27 (td, J=13.1, 5.9 Hz, 2H), 7.53-7.49 (m, 2H), 7.46-7.40 (m,2H), 7.36-7.25 (m, 4H), 6.64 (t, J=12.4 Hz, 1H), 6.33 (d, J=13.7 Hz,2H), 5.91 (dt, J=14.9, 7.2 Hz, 1H), 5.44 (dd, J=15.6, 5.1 Hz, 1H), 5.00(t, J=3.8 Hz, 1H), 4.59 (d, J=5.1 Hz, 1H), 4.26 (dt, J=10.8, 7.0 Hz,4H), 3.92 (dt, J=51.0, 7.0 Hz, 4H), 3.16 (s, 6H), 2.65 (q, J=6.7 Hz,2H), 2.21 (q, J=7.0 Hz, 2H), 1.74 (d, J=3.2 Hz, 12H). ¹³C NMR (101 MHz,CD₃OD) δ 173.5, 173.4, 154.1, 142.2, 142.1, 141.2, 141.2, 130.4, 130.0,128.3, 128.3, 125.3, 124.8, 124.8, 122.0, 122.0, 110.9, 110.7, 103.5,103.1, 103.0, 101.6, 64.7, 51.9, 49.2, 49.2, 42.5, 38.6, 30.2, 30.0,26.6, 26.3. HRMS (ESI) calculated for C₃₇H₄₇N₂O₄ (M⁺) 583.3530, observed583.3536.

(4): In a sealed vial, aqueous H₂SO₄ (3.5 M, 1.3 mL) was added to a darkblue solution of 3 (37 mg, 0.059 mmol) in CHCl₃ (5.0 mL). This biphasicmixture (1:4 20% H₂SO₄/CHCl₃) was stirred at 70° C. for 3 hours, atwhich time LC/MS analysis revealed complete consumption of startingmaterial. The resulting solution was quenched with saturated aqueousNaHCO₃ (20 mL), extracted with CH₂Cl₂ (4×10 mL), and dried over Na₂SO₄.The solvent was evaporated under reduced pressure and the blue-greenresidue was purified with normal phase chromatography (12 g silica goldcolumn, 0-15% MeOH/CH₂Cl₂). The resulting product was dissolved in 1:1aqueous NaI (1.0 M, 15 mL) and CH₂Cl₂ (15 mL), and stirred vigorously atroom temperature for 5 hours. The biphasic mixture was separated,extracted with CH₂Cl₂ (3×10 mL CH₂Cl₂), and dried in vacuo. Theresulting residue was eluted from a pipet packed with silica (10%MeOH/90% CH₂Cl₂) and evaporated under reduced pressure to afford 4 (8.64mg, 0.014 mmol, 24%) as a blue solid.

Compound 4 was a single diastereomer with syn-syn ring junctionstereochemistry assigned by NMR analysis (FIG. 4). The calculations wereperformed using Spartan '14 (Wavefunction, Inc., Irvine, Calif.). Exceptfor molecular mechanics and semi-empirical models, the calculationmethods used in Spartan have been documented in Shao et al. (Phys. Chem.Chem. Phys. 2016, 8:3172). Conformational distribution was performed atthe MMFF level. The three lowest energy conformers (within 20 kJ/mol ofthe lowest energy) in the previous step were subjected to geometryoptimization using B3LYP/6-31G(d), EtOH solvent. (1 for syn-syn, 2 forsyn-anti, 3 for anti-syn, 3 for anti-anti). The resulting minima areshown in FIG. 4. All three methine protons which are located at the ringjunctions (positions 12, 14, and 15) exhibited ID-NOESY interactionswith the upfield axial α-nitrogen protons (3.88 (tq, J=13.3, 4.5 Hz,2H)—position 10 and 17), consistent with the syn, syn diastereomer.Furthermore, none of these protons displayed an NOE to the downfieldequatorial α-nitrogen protons (4.40-4.24 (m, 2H)—positions 10 and 17),which would be expected for at least one methine proton in any otherdiastereomeric structure. Also, consistent with the formation of thesyn-syn diastereomer, the α-oxygen protons (positions 14 and 15)displayed nearly identical coupling constants, indicating these protonsare in similar ring systems.

(4): To a solution of 3 (11.0 mg, 0.018 mmol, 1 eq) in anhydrous CH₂Cl₂(1.8 mL, 0.01 M), degassed under argon and cooled to −78° C. (dry iceand acetone bath), BBr₃ (0.21 mL of a 1.0 M CH₂Cl₂ solution, 0.21 mmol,12 eq) was slowly added. The reaction color rapidly transitioned fromdark blue to red-brown upon BBr₃ addition. After stirring for 2 hoursunder argon at −78° C., the reaction was quenched with the addition ofaqueous NaHCO₃ (0.5 M, 5 mL) and the blue color rapidly returned. Oncewarmed to room temperature, additional CH₂Cl₂ (10 mL) was provided andthe biphasic mixture was stirred vigorously for 45 min. The lightgreen/yellow aqueous layer was then extracted with CH₂Cl₂ (3×5 mL), andthe combined dark blue organic solution was dried over Na₂SO₄ andevaporated under reduced pressure. This blue residue contained adiastereomeric mixture of 4 and a second compound (obtained in avariable ratio of −1:1 to −2:1). The inseparable mixture exhibited asingle [M]⁺ ion signal, complex NMR signals in the dihydropyran region,and a single far-red UV-vis absorbance maximum. The mixture wasequilibrated to homogenous compound 4 as follows. The mixture wasdissolved in MeOH (0.9 mL) and aqueous HCl (0.3 M, 2.7 mL), and stirredat 60° C. for 20 min. After cooling to room temperature, aqueous NaHCO₃(0.5 M, 5 mL) and CH₂Cl₂ (10 mL) were added to form a biphasic mixture,which was stirred vigorously for 30 min. The light green/yellow aqueouslayer was extracted with CH₂Cl₂ (10×5 mL), and the combined organicsolution was dried over Na₂SO₄, and concentrated under reduced pressure.The blue residue was dissolved in CH₂Cl₂ (15 mL) and aqueous NaI (0.5 M,10 mL) and stirred vigorously for 13 hours at room temperature. Thebiphasic mixture was separated, extracted with CH₂Cl₂ (2×10 mL), anddried over Na₂SO₄. After drying in vacuo, the residue was purified bynormal phase chromatography (4 g silica gold column, 0-10% MeOH/CH₂Cl₂)to afford a blue solid of 4 as a single diastereomer (5.7 mg, 0.0095mmol, 53%). ¹H NMR (400 MHz, CD₃OD) δ 7.93 (s, 1H), 7.86 (s, 1H),7.57-7.36 (m, 4H), 7.37-7.16 (m, 4H), 4.67 (dd, J=11.5, 5.0 Hz, 1H),4.61 (dd, J=11.4, 5.0 Hz, 1H), 4.40-4.24 (m, 2H), 3.88 (tq, J=13.3, 4.5Hz, 2H), 2.84 (tt, J=12.3, 4.1 Hz, 1H), 2.58 (dt, J=11.7, 4.5 Hz, 1H),2.49 (dt, J=11.7, 4.5 Hz, 1H), 2.41 (dt, J=13.3, 4.2 Hz, 1H), 2.01 (qd,J=12.6, 5.4 Hz, 1H), 1.82 (dd, J=12.6, 5.0 Hz, 1H), 1.78-1.74 (m, 12H),1.48 (q, J=11.8 Hz, 1H). ¹³C NMR (126 MHz, CD₃OD) δ 169.2, 165.8, 143.3,142.0, 142.0, 141.5, 141.1, 140.3, 128.7, 128.3, 128.3, 125.3, 124.5,121.9, 121.9, 114.0, 111.4, 110.2, 109.6, 72.2, 70.2, 49.1, 48.3, 42.9,40.7, 35.1, 31.1, 27.2, 26.7, 26.7, 26.6, 26.1, 26.0. HRMS (ESI)calculated for C₃₃H₃₅N₂O (M⁺) 475.2737, observed 475.2744. Computationalanalysis indicates the lowest energy diastereomer containing ananti-ring junction was diastereomeric to compound 4 adjacent to C5′ ofthe polyene (FIG. 5). It was noted that simply subjecting compound 3 tothe MeOH:HCl equilibration conditions provided only trace quantities ofcompound 4.

(S6): The following was placed in three separate sealed vials:4-hydrazinyl-benzoic acid (S5, 610 mg, 4.0 mmol, 1.0 eq),3-methyl-2-butanone (430 μL, 4.0 mmol, 1.0 eq), KHSO₄ (1.5 g, 12 mmol,3.0 eq) and MeOH (15 mL). Each vial was heated to 160° C. for 2 hours bymicrowave. The combined contents of three vials were separated betweensaturated aqueous NaHCO₃ (100 mL) and CH₂Cl₂ (2×100 mL), dried (NaHSO₄)and concentrated under reduced pressure. The resulting residue waspurified by normal phase chromatography (40 g silica column, 30-100%ethyl acetate/hexanes) to provide S6 (1.4 g, 6.3 mmol, 53%) as a tanoil. ¹H NMR (400 MHz, CDCl₃) δ 8.06 (dd, J=8.1, 1.7 Hz, 1H), 7.99 (d,J=1.6 Hz, 1H), 7.58 (d, J=8.1 Hz, 1H), 3.95 (s, 3H), 2.34 (s, 3H), 1.36(s, 6H). ¹³C (126 MHz, CDCl₃) δ 191.7, 167.3, 157.7, 145.7, 130.1,126.9, 122.7, 119.6, 53.9, 52.1, 22.9, 15.7. HRMS (ESI) calculated forC₁₃H₆NO₂ (MH⁺) calculated 218.1176, observed 218.1173.

(S7): In a sealed vessel, S6 (6.0 g, 28 mmol, 1.0 eq) was combined with2-(2-bromoethyl)-1,3-dioxolane (5.0 mL, 41 mmol, 1.5 eq), NaHCO₃ (4.6 g,55 mmol, 2.0 eq), and NaI (6.2 g, 41 mmol, 1.5 eq) in MeCN (80 mL). Thereaction was stirred at 95° C. for 14.5 hours as the solution colortransitioned from orange to brown. The crude product was filtered overcelite and concentrated under reduced pressure and the residue waspurified by reversed-phase column chromatography (150 g C18 gold column,0-80% MeCN/H₂O) to afford S7 (2.8 g, 8.9 mmol, 32%) as a dark green oil.¹H NMR (400 MHz, CDCl₃) δ 7.90 (dd, J=8.3, 1.8 Hz, 1H), 7.75 (d, J=1.7Hz, 1H), 6.62 (d, J=8.3 Hz, 1H), 4.93 (t, J=4.5 Hz, 1H), 4.09 (d, J=2.3Hz, 1H), 4.04-3.84 (m, 8H), 3.72 (t, J=7.2 Hz, 2H), 2.03 (td, J=7.5, 4.5Hz, 2H), 1.36 (s, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 167.6, 160.7, 149.8,137.6, 131.1, 123.3, 120.2, 104.7, 102.5, 76.4, 65.1, 51.79, 43.81,37.39, 30.23, 30.01. HRMS (ESI) calculated for C18H₂₃NO₄ (MH⁺) 318.1700,observed 318.1695.

(S9): To a heterogenous mixture of S8 (4.6 g, 19 mmol, 1.0 eq) in MeCN(50 mL, 0.38 M) at room temperature was slowly added freshly-preparedneat 3-butenyl triflate (S2, 5.1 g, 25 mmol, 1.3 eq). After 15 min, theresulting dark mixture was treated with saturated aqueous NaHCO₃ (50mL). The resulting mixture was filtered through celite and the volume ofthe resulting solution was reduced on a rotary evaporator (˜50 mL). Thissolution was purified with reversed phase chromatography (150 g C18Aqgold column, 0-15% MeCN/H₂O) to provide clean S9 (2.2 g, 7.4 mmol, 40%)as a pink solid. ¹H NMR (400 MHz, DMSO-d) 7.35 (dd, J=8.0, 1.7 Hz, 1H),7.33 (d, J=1.5 Hz, 1H), 6.57 (d, J=8.1 Hz, 1H), 5.83 (ddt, J=17.1, 10.2,7.0 Hz, 1H), 5.06 (dq, J=17.2, 1.4 Hz, 1H), 4.98 (dd, J=10.1, 2.1 Hz,1H), 3.94 (d, J=1.8 Hz, 1H), 3.90 (d, J=1.8 Hz, 1H), 3.60 (t, J=7.1 Hz,2H), 2.31 (q, J=6.9 Hz, 2H), 1.26 (s, 6H). ¹³C NMR (126 MHz, DMSO-d₆) δ160.9, 145.9, 139.5, 136.3, 136.1, 125.9, 120.0, 117.3, 104.4, 75.2,44.0, 41.2, 30.5, 30.2. HRMS (ESI) calculated for C₁₅H₁₉NO₃S (MH⁺)294.1158, observed 294.1155.

(9): To a solution of S9 (2.4 g, 7.7 mmol, 1.5 eq), S7 (1.5 g, 5.1 mmol,1 eq), malonaldehyde bis(phenylimine) monohydrochloride (1.6 g, 6.1mmol, 1.2 eq), and Et₃N (3.6 mL, 26 mmol, 5 eq) in MeOH (26 mL) wasadded acetic anhydride (1.0 mL, 10 mmol, 2 eq). While stirring at roomtemperature, additional acetic anhydride (1.0 mL, 10 mmol, 2 eq) wasadded over the first 1.5 hours of reaction approximately every 30 min.to total 3.9 mL (41 mmol, 8 eq). During the reaction, the solution colortransitioned from red to green to purple and finally to dark blue. After4 hours of total reaction time, LC/MS analysis revealed completeconsumption of S7, and a mixture of the 3 possible cyanine products inan approximately 2:1:1 ratio of the unsymmetrical desired product to theundesired symmetrical cyanines. The dark blue solution was precipitatedin 2:1 diethyl ether/hexanes (180 mL), centrifuged (6800 rpm, 8 min),and the supernatant was removed. This trituration was performed threetimes, and after drying in vacuo the dark blue residue was purified withreverse-phase chromatography (150 g gold C18 column, 0-80% MeCN/H₂O) toprovide 5 (0.72 g, 1.1 mmol, 22%) as a dark blue solid. ¹H NMR (400 MHz,CD₃OD) δ 8.35 (dt, J=17.0, 13.0 Hz, 2H), 8.14-8.06 (m, 2H), 7.97-7.89(m, 2H), 7.44 (d, J=8.3 Hz, 1H), 7.34 (d, J=8.3 Hz, 1H), 6.72 (t, J=12.4Hz, 1H), 6.48 (d, J=13.9 Hz, 1H), 6.34 (d, J=13.4 Hz, 1H), 5.90 (ddt,J=17.2, 10.2, 7.1 Hz, 1H), 5.10-5.02 (m, 2H), 4.99 (t, J=3.8 Hz, 1H),4.30 (t, J=6.9 Hz, 2H), 4.26 (t, J=7.0 Hz, 2H), 3.98-3.84 (m, 7H), 2.64(q, J=7.1 Hz, 2H), 2.21 (td, J=7.0, 3.8 Hz, 2H), 1.77 (d, J=5.6 Hz,12H). ¹³C NMR (126 MHz, CD₃OD) δ 175.8, 172.6, 166.6, 155.8, 154.2,146.4, 143.2, 142.7, 141.4, 141.1, 133.4, 130.5, 126.7, 125.8, 123.0,120.0, 117.8, 111.1, 110.0, 105.5, 103.6, 101.6, 64.7, 51.3, 49.7, 48.6,48.2, 43.1, 38.5, 31.7, 30.0, 26.4. HRMS (ESI) calculated forC₃₆H₄₃N₂O₇S (MH⁺) 647.2785, observed 647.2776.

(10): To a solution of 5 (120 mg, 0.19 mmol, 1.0 eq) and Hoyveyda-Grubbscatalyst 2^(nd) generation (58 mg, 0.093 mmol, 0.5 eq) in anhydrousCH₂Cl₂ (9.3 mL) was added acrolein dimethyl acetal (2, 110 μL, 0.95mmol, 5.0 eq) under argon. The blue solution was stirred at roomtemperature for 5 hours with a closed reflux condenser under argon andstatic vacuum, which was reapplied approximately every 30 min. Theresulting blue mixture was precipitated in 1:1 diethyl ether/hexanes (45mL), centrifuged (7500 rpm, 5 min.), and decanted. After repeating thisprecipitation, the pellet was dried under high vacuum to afford 6 (130mg, 0.18 mmol, 97%) as a dark blue solid that was used without furtherpurification. ¹H NMR (500 MHz, CD₃OD) δ 8.36 (q, J=13.7 Hz, 2H),8.13-8.08 (m, 2H), 7.95-7.90 (m, 2H), 7.46-7.41 (m, 1H), 7.35 (dd,J=8.3, 2.2 Hz, 1H), 6.72 (t, J=12.4 Hz, 1H), 6.48 (d, J=13.8 Hz, 1H),6.35 (d, J=13.4 Hz, 1H), 5.90 (ddd, J=14.6, 7.6, 6.5 Hz, 1H), 5.49-5.37(m, 1H), 4.99 (t, J=3.8 Hz, 1H), 4.58 (dd, J=5.3, 1.0 Hz, 1H), 4.32 (t,J=6.9 Hz, 2H), 4.26 (t, J=7.1 Hz, 2H), 4.01-3.79 (m, 7H), 3.37 (s, 3H),2.66 (td, J=8.0, 7.4, 5.3 Hz, 2H), 2.21 (td, J=7.0, 4.0 Hz, 2H),1.81-1.75 (m, 12H). ¹³C NMR (126 MHz, MeOD) δ 175.7, 172.7, 166.6,155.7, 154.2, 146.3, 143.3, 142.7, 141.4, 141.2, 130.8, 130.6, 129.7,126.7, 126.7, 125.9, 123.0, 120.0, 111.1, 110.1, 105.6, 103.7, 102.8,101.6, 64.7, 51.3, 49.6, 48.6, 48.4, 48.2, 43.1, 38.6, 30.0, 26.3. HRMS(ESI) calculated for C₃₉H₄₈N₂O₉S (MH⁺) 721.3153, observed 721.3144.

(S10): A dark blue solution of 6 (0.13 g, 0.18 mmol, 1 eq) in CH₂Cl₂ (12mL, 0.015 M) was cooled to −78° C. (dry ice and acetone bath) anddegassed under argon. BBr₃ (2.1 mL of a 1.0 M CH₂Cl₂ solution, 2.1 mmol,12 eq) was added slowly and the reaction color transitioned tored-brown. The reaction was stirred under argon with the temperaturemaintained at −78° C. over 4 hours at which time the reaction wasquenched with the addition of H₂O (10 mL) and immediately returned to ablue color. LC/MS analysis revealed complete consumption of startingmaterial. After warming to room temperature, the organic solvent wasevaporated under reduced pressure. The remaining blue aqueous solutionwas purified by reverse-phase chromatography (30 g C18 gold column,0-60% MeCN/H₂O) to yield a diastereomeric mixture of compound S10 (57mg, 0.094 mmol, 53%) as a dark blue solid. HRMS (ESI) calculated forC₃₅H₃₆N₂O₆S (MH⁺) 613.2367, observed 613.2360.

(7): A diastereomeric mixture of S10 (43 mg, 0.070 mmol) was dissolvedin MeOH (5.8 mL) and aqueous HCl (0.30 M, 17 mL). This reaction mixturewas heated to 60° C. for 7 hours over which time the color transitionedfrom dark blue to a green-blue color. After cooling to room temperature,the reaction was dried under reduced pressure to yield a singlediastereomer of S10 as confirmed by NMR analysis. This crude solid ofthe equilibrated methyl ester intermediate was redissolved in MeOH (3.5mL) and aqueous LiOH (2.0 M, 3.5 mL). The resulting blue solution wasstirred at room temperature for 2.5 hours, at which time LC/MS analysisrevealed complete conversion to 7. Saturated aqueous NaHCO₃ (3.0 mL) wasadded to quench the reaction. After the removal of MeOH in vacuo, thecrude aqueous mixture was purified by reverse-phase chromatography (30 gC18 gold column, 0-70% MeCN with 0.05% formic acid/H₂O with 0.05% formicacid), to afford 7 (34 mg, 0.057 mmol, 82%) as a blue solid. ¹H NMR (500MHz, DMSO-d₆) δ 8.08 (s, 1H), 8.04 (s, 1H), 7.99 (s, 1H), 7.95 (d, J=8.2Hz, 1H), 7.89 (s, 1H), 7.69 (d, J=8.1 Hz, 1H), 7.36 (d, J=8.2 Hz, 1H),7.23 (d, J=8.3 Hz, 1H), 4.57 (dd, J=11.4, 4.7 Hz, 1H), 4.51 (dd, J=11.3,4.6 Hz, 1H), 4.34 (d, J=10.3 Hz, 1H), 4.24 (d, J=9.5 Hz, 1H), 3.89 (t,J=11.7 Hz, 1H), 3.80 (t, J=12.9 Hz, 1H), 2.75 (t, J=12.4 Hz, 1H), 2.43(d, J=12.2 Hz, 1H), 2.35 (dd, J=7.0, 4.5 Hz, 1H), 2.28 (d, J=12.9 Hz,1H), 1.88 (dd, J=12.2, 5.0 Hz, 1H), 1.77-1.62 (m, 13H), 1.34 (q, J=11.8Hz, 1H). ¹³C NMR (126 MHz, DMSO-d6)δ 169.4, 166.3, 162.4, 145.1, 143.9,142.9, 140.4, 140.0, 139.3, 138.6, 129.2, 128.4, 124.9, 122.1, 118.8,114.4, 109.7, 109.3, 107.9, 70.2, 68.4, 48.2, 46.3, 42.1, 39.4, 33.5,29.2, 26.3, 25.9, 25.6, 25.4. HRMS (ESI) calculated for C₃₅H₃₆N₂O₆S(MH⁺) 599.2210, observed 599.2204. Compound 7 was a single diastereomerwith syn-syn ring junction stereochemistry assigned by NMR analysis(FIG. 6). All three methine protons which are located at the ringjunctions (positions 12, 14, and 15) exhibited ID-NOESY interactionswith the upfield axial α-nitrogen protons (3.88 (tq, J=13.3, 4.5 Hz,2H)—position 10 and 17), consistent with the syn, syn diastereomer.Furthermore, none of these protons displayed an NOE to the downfieldequatorial α-nitrogen protons (4.40-4.24 (m, 2H)—positions 10 and 17),which would be expected for at least one methine proton in any otherdiastereomeric structure. Also, consistent with the formation of thesyn-syn diastereomer, the α-oxygen protons (positions 14 and 15)displayed nearly identical coupling constants, indicating these protonsare in similar ring systems.

(S11): To a solution of 7 (6.0 mg, 0.010 mmol, 1 eq) in DMF (0.20 mL,0.05 M) was added TSTU (4.5 mg, 0.015 mmol, 1.5 eq) and DIPEA (1.8 μL,0.020 mmol, 2 eq). This blue solution was stirred at room temperaturefor 1.5 hours at which time LC/MS analysis revealed complete conversionto S11. The mixture was then precipitated in ether (14 mL), centrifuged(6000 rpm, 5 min), and the supernatant was decanted. After repeatingthis ether wash, the dark blue solid was dried in vacuo to afford S11(6.9 mg, 0.0098 mmol, 98%), which was used without further purification.

(8): To a solution of S11 (3.0 mg, 0.0043 mmol, 1 eq) in DMF (0.15 mL,0.03 M) was added NH₂—PEGs-COOH (2.3 mg, 0.0053 mmol, 1.2 eq) and DIPEA(N,N-diisopropylethylamine, 3.8 μL, 0.022 mmol, 5 eq). This bluesolution was stirred at room temperature for 1.5 hours at which timeLC/MS analysis revealed complete consumption of S12. The resultingsolution was precipitated in 1:1 ether and hexanes (14 mL), centrifuged(4000 rpm, 5 min), and the supernatant was removed. This trituration wasrepeated, and the resulting blue residue was purified by reverse-phasechromatography (5.5 g C18Aq gold column, 0-70% MeCN/H₂O with 0.05%formic acid) to afford 8 (2.3 mg, 0.0023 mmol, 54%) as a blue solid.HRMS (ESI) calculated for C₅₃H₇₁N₃O₁₅S (MH⁺⁾ 1022.4679, observed1022.4679.

(S12): To a solution of 8 (3.4 mg, 0.0033 mmol, 1 eq) in DMF (0.17 mL,0.02 M) was added TSTU (1.5 mg, 0.0050 mmol, 1.5 eq) and DIPEA (1.7 μL,0.010 mmol, 3 eq). This blue reaction mixture was stirred at roomtemperature for 2 hours at which time LC/MS analysis revealed completeconversion. The solution was precipitated in 1:1 ether/hexanes (14 mL),centrifuged (6500 rpm, 5 min), and the supernatant decanted. Afterrepeating this precipitation, the blue residue was dried under reducedpressure to yield S12 (3.4 mg, 0.0030 mmol, 91%), which was used withoutfurther purification.

(9): To a solution of S12 (0.98 mg, 8.8×10⁻⁴ mmol, 1 eq) in DMSO (0.12μL, 0.008 M) was added aminophalloidin tosylate (0.80 mg, 8.8×10⁻⁴ mmol,1 eq) and DIPEA (0.73 μL, 0.0042 mmol, 5 eq). This solution was stirredat room temperature for 1 hour at which time LC/MS analysis revealedconsumption of aminophalloidin tosylate. The blue reaction mixture wasprecipitated in 1.5:1 ether/hexanes (1.4 mL), centrifuged (6000 rpm, 30sec.), and the supernatant was removed. This trituration was performed atotal of 3 times. After drying under reduced pressure, the blue residuewas purified by reverse-phase preparative HPLC (20-95% MeCN/H₂O with0.1% formic acid) and lyophilized to yield 9 (1.2 mg, 6.6×10⁴ mmol, 76%)as a blue solid. HRMS (ESI) calculated for C₈₈H₁₁₈N₁₂O₂₄S₂ ([M+2H⁺]⁺²)896.3984, observed 896.3971.

Example 2 Exemplary Syntheses for Conformationally RestrictedHeptamethine Cyanine Fluorophores

The indolenine and bis vinylogous amide were combined in 10:1 EtOH/AcOHand heated to 70° C. After 2 h, the mixture was extracted with CH₂Cl₂and saturated sodium bicarbonate. The organic layers were dried withsodium sulfate, concentrated, and then purified with normal phasechromatography to provide the product.

To a solution of the ketone in CH₂Cl₂ was added 10 equiv. of acroleindimethyl acetal and 0.4 equiv. of HG-2. The solution was maintained atroom temperature for 18 h, concentrated and purified by normal phasechromatography to provide the product.

A solution of the ketone in THF was treated with 1 N HCl. After 30 min,the mixture was extracted with CH₂Cl₂ and saturated sodium bicarbonate.The organic layers were dried with sodium sulfate, concentrated, andthen used in the next step without purification.

The ketone, CH₂Cl₂, and pyridine were cooled to −78° C. and Tf₂O(trifluoromethanesulfonic anhydride) was added. The solution was allowedto warm to room temperature, and extracted with CH₂Cl₂ and saturatedsodium bicarbonate. Purification with normal phase chromatographyprovided the product.

The triflate, boronic acid, and Pd(PPh₃)₄ were dissolved in 1:1iPrOH:H₂O and heated to 90° C. After 18 h, the mixture was extractedwith CH₂Cl₂ and saturated sodium bicarbonate. The organic layers weredried with sodium sulfate, concentrated, and then purified with normalphase chromatography to provide the product.

Example 3 Characterization of Conformationally Restricted CyanineFluorophores

The spectroscopic properties of conformationally restricted compounds 4and 7 were compared to those of unrestricted compound 10.

TABLE 1 Spectroscopic Properties λ_(max) ε λ_(em) τ Cpd (nm) (M⁻¹cm⁻¹⁾(nm) Φ_(F) (ns) 10^(a) 638 214,000 657 0.15 0.7  4^(a) 662 206,000 6770.69 2.5  7  670^(b) 190,000  683^(b) 0.55^(b) 1.7^(c) ^(a)in methanol;^(b)in pH 7.4 PBS; ^(c)in H₂O

Compounds 4 and 7 exhibited the characteristic features ofconformational restriction. The quantum yield is increased from 0.15(MeOH) with compound 10 to 0.69 (MeOH) and 0.55 (PBS) with compounds 4and 7, respectively (Table 1). This occurs with a shift in λ_(max) ofapproximately nm in both cases. Both fluorescence lifetime and quantumyield are largely solvent viscosity insensitive, unlike with theconventional pentamethine cyanine 10 that is subject tophotoisomerization (Table 2).

TABLE 2 Fluorescence Lifetime (τ) and Quantum Yield (Φ_(F)) of Compounds4 and 10 4 10 Solvent τ (ns) Φ_(F) τ (ns) Φ_(F) MeCN 2.8 0.92 0.7 0.13MeOH* 2.5 0.69 0.7 0.15 EtOH 2.5 0.68 0.8 0.18 Acetone 2.9 0.90 0.8 0.19Glycerol 2.3 0.66 1.8 0.24 *Reference Φ_(F) by Integrating Sphere -other Φ_(F) values calculated by relative method

Moreover, also unlike with compound 10 (circles), the emission ofcompound 4 (squares) is insensitive to temperature (FIG. 7). This isalso due to photoisomerization in compound 10, which becomes moreefficient at higher temperatures. The substantially longer lifetimes ofcompounds 4 and 7 relative to the unrestrained cyanine 10 point tosignificant potential for fluorescence lifetime imaging microscopy(FLIM).

Example 4 Single-Molecule Localization Microscopy with ConformationallyRestricted Cyanine Fluorophores

A central feature of single-molecule localization microscopy (SMLM) isthe photoactivation or conversion of fluorophores between fluorescentand nonfluorescent states. Three modes of cyanine reactivity have beenapplied in this context: reversible formation of (1) thiol- and (2)phosphine-polyene adducts, as well as (3) sequential reduction/oxidationof the imine-like C₂—N double bond.

Compounds 4, 7, and 10 of Example 3 were evaluated. Most strikingly, theUV-light induced regeneration of compound 4 following NaBH₄ reductionwas dramatically enhanced relative to unrestrained cyanines. Reductionof compound 4 with 2.0 equiv. of NaBH₄ (2.5 mM in 1:1 DMSO:MeOH)followed by photolysis of a 20 μM solution in 4:1 PBS (50 mM, pH7.4):DMSO with UV light (365 nm, 5 mW/cm²) provided 38% maximal cyanineabsorbance recovery with compound 4 (squares) after 5 min, but only a 6%maximal recovery after 30 minutes with compound 10 (circles) (FIG. 8).Absorbance was measured at λ_(max) (compound 4 at 640 nm and compound 10at 660 nm as a function of time of 365 nm irradiation (5 mW/cm²).

The formation of phosphine and thiol adducts in chemical and singlemolecule imaging contexts was also evaluated. TCEP(tris(2-carboxyethyl)phosphine) was add to compound 7 and AF647 (AlexaFluor 647, available through ThermoFisher Scientific) (10 μM each)(Vaughan et al., JACS 2013, 135(4): 1197-1200). Absorbance at λ_(max)(compound 7 at 670 nm and AF647 at 650 nm) as a function of TCEPconcentration in Tris (0.20 M, pH 9.0). Compound 7 (circles) appearedresistant to the formation of polyene-heteroatom adducts (FIG. 9).

Example 5 Binding and Visualization of a Conformationally RestrictedPhalloidin Conjugate

The phalloidin conjugate 9 of Example 3 was applied to visualizecellular F-actin in initial wide-field studies. These efforts includedcomparisons to the commercially available Alexa Fluor 647-phalloidinconjugate (AF647-phalloidin), which has been used extensively. Reductionand UV-activation (370 nm) of compound 9 provided dramatically improvedrecovery relative to AF647-phalloidin. Photostability of compound 9 wasnearly indistinguishable from that of AF647-phalloidin.

The phalloidin conjugate 9 was evaluated with 3D PALM-likesuper-resolution imaging utilizing a biplane imaging scheme (BP) (Ram etal., J., Biophys J2008, 95:6025) in combination with TRABI (Franke etal. Nat Methods 2017, 14:41) to simultaneously precisely quantify singlemolecule intensities and perform TRABI-BP imaging. Labeling andreduction (26 mM NaBH₄) with subsequent imaging in non-degassedphosphate buffered saline (PBS) provided high quality 3D super-resolvedimages of the actin cytoskeleton in a U2OS (human bone osteosarcomaepithelial) cell (FIG. 10). An average of 5181 photons per frame(median) were detected from single activated dyes, while trackingemitters that are active in consecutive frames yielded a conflatedphoton count of 6961 (median) before photobleaching or conversion to anonfluorescent form. This corresponds to experimentally measuredlocalization precisions of 5-7 nm laterally and ˜20 nm axially.Excitation with either 640 or 660 nm can be employed, with the laterproviding somewhat improved photon yield. While the UV-laser canaccelerate the recovery, the photoactivation rate obtained using solelythe excitation laser (either 640 or 660) was sufficient to generate anemitter density suitable for SMLM. In SMLM experiments recovery of thereduced state proceeded almost quantitatively if 405 nm light wasapplied only for very short time periods. By contrast, whenAF647-phalloidin was subjected to the reduction/recovery sequence, noreconstruction could be obtained. The images obtained with compound 9using the reductive method were compared with AF647 under standarddSTORM buffer conditions. Conjugate 9 gave similar, if slightlyimproved, photon counts relative to AF647-phalloidin (9: 3721 per frame,5107 tracked, AF647: 3422 per frame, 3737 tracked) and localizationprecisions of 5.2 & 5.9 nm respectively (FIGS. 11-13). FIG. 11 showslateral and axial localization precisions calculated from the image ofFIG. 10. FIG. 12 is a bar graph showing a comparison of single moleculephoton intensities regarding single frame (black) and tracked (grey)median values of data illustrated for compound 9_(3D) (left) andcomparable measurements in 2D imaging modes of compound 9 (center) andAF647 (right) in standard dSTORM photoswitching buffer. FIG. 13 is a bargraph showing experimentally determined lateral localization precisionsof compound 9_(3D) (6.9 nm),compound 9_(2D) (5.2 nm) and AF6472D (5.9nm).

Example 6 Tumor Visualization with Conformationally Restricted CyanineFluorophores

A subject having a tumor is identified and selected for treatment. Thesubject may be selected based on a clinical presentation and/or byperforming tests to demonstrate presence of a tumor.

The subject is treated by administering a compound according to FormulaI, a pharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition thereof at a dose determined by a clinician to be effective.The compound is administered by any suitable means, such as intravenousor subcutaneous injection. In some instances, the compound is injecteddirectly into the tumor.

Visualization may be performed after a period of time sufficient toallow binding of the compound to the tumor. For example, irradiation maybe performed several hours to several days after administration of thecompound, such as from 1-7 days after administration of the compound.The administered compound is irradiated by targeted application of aneffective quantity of light having a wavelength and a selected intensitysuitable for inducing fluorescence of the cyanine fluorophore to atargeted portion of the subject, thereby exciting the cyaninefluorophore. Advantageously, the portion of the subject targeted forirradiation is proximate the tumor. Fluorescence of the compound isdetected by any suitable method known to a person of ordinary skill inthe art of fluorescence imaging. Fluorescence-guided surgery is used todetermine the location and extent of tissue excision.

In some cases, the subject is suspected of having a tumor and presenceof a tumor is confirmed by administering the compound to the subject andmonitoring the compound's fluorescence at a suspected tumor site.Accumulation of the compound and fluorescence at the suspected tumorsite diagnoses presence of a tumor.

With reference to FIG. 14, a subject 100 with a tumor 110 may be treatedwith a compound according to Formula I that comprises an antibody orligand capable of recognizing and binding to an antigen or receptor on atumor cell surface. In the example shown in FIG. 14, the compound 120 isadministered via intravenous injection. A period of time is allowed toelapse during which the compound preferentially accumulates at the tumorsite as the antibody or ligand moiety binds to the tumor. A targetportion of the subject subsequently is selectively irradiated with aneffective amount of far-red or NIR light energy of a desired wavelengthusing an external light applicator 130. The light applicator 130 appliesthe light to a target area limited to the region of the tumor 110,thereby producing fluorescence of the compound. The tumor is visualizedby detecting the fluorescence.

A therapeutically effective amount of a second agent may beco-administered with the compound according to Formula I or saltthereof. The compound (or salt thereof) and the second agent may beadministered either separately or together in a single composition. Thesecond agent may be administered by the same route or a different route.If administered concurrently, the compound (or salt thereof) and thesecond agent may be combined in a single pharmaceutical composition ormay be administered concurrently as two pharmaceutical compositions. Thesecond agent may be, for example, an anti-tumor agent or an angiogenesisinhibitor.

Example 6 Synthesis of a Bis-Sulfonated Conformationally RestrictedCyanine Fluorophore

The synthesis of conformational restricted cyanine dye 17 is describedin FIG. 15. Starting from the racemic methyl 6-methyl-7-oxooctanoate 11,a Fisher indole synthesis was carried out with4-hydrazineylbenzenesulfonic acid to produce 12 in 76% yield. Thesubsequent N-alkylation was performed in the presence of2-(2-iodoethyl)-1,3-dioxolane at 95° C. to provide the product 3 in 51%yield. The cyanine skeleton was formed by reacting three moieties:compound 13,1-(but-3-en-1-yl)-2,3,3-trimethyl-3H-indol-1-ium-5-sulfonate potassiumsalt, and N-((1E,3E)-3-(phenylimino)prop-1-en-1-yl)aniline to afford thedesired product 14 in 24% yield. Cross metathesis was performed withacrolein dimethyl acetal under Hoyveyda-Grubbs catalyst and Bu₄NBr toproduce 15. The key intramolecular Michael addition associatedannulation cascade proceeded in the presence of BBr₃ and Bu₄NBr to yielddiastereomeric mixtures, which was converted to two diastereomers 16under the equilibration condition of HCl/MeOH at 60° C. The yield was41% in the three steps. Hydrolysis of methyl ester with LiOH gave thefinal product 17 in 43% yield after the reverse phase C-18 columnpurification.

Experimental Details Sodium3-(5-methoxy-5-oxopentyl)-2,3-dimethyl-3H-indole5-sulfonate (12)

Methyl 6-methyl-7-oxooctanoate (1.86 g, 10 mmol) was added to a stirredsolution of p-hydrazinobenzenesulfonic acid (2.24 g, 10 mmol) in aceticacid (6 mL). The solution was heated to reflux for 4 h, then cooled toroom temperature. The solvents were evaporated. Na₂CO₃ (1.06 g, 10 mmol)was added to the residue dissolved in methanol (15 mL). The resultingmixture was stirred at room temperature for 15 h. The solvents wereevaporated, and the residue was purified by reverse phase columnchromatography to provide 12 (2.72 g, 7.6 mmol, 76%). ¹H NMR (400 MHz,CD₃OD): δ 7.84-7.80 (m, 2H), 7.46 (d, 1H, J=7.8 Hz), 3.57 (s, 3H), 2.28(s, 3H), 2.16 (dt, 2H, J=7.4, 2.3 Hz), 2.04-1.84 (m, 2H), 1.50-1.41 (m,2H), 1.34 (s, 3H), 0.74-0.56 (m, 2H). LC-MS (ESI) 340 (M+).

1-(2-(1,3-Dioxolan-2-yl)ethyl)-3-(5-methoxy-5-oxopentyl)-2,3-dimethyl-3H-indol-1-ium-5-sulfonate,sodium salt (13)

In a sealed vessel, compound 12 (2.25 g, 6.23 mmol) was combined with2-(2-bromoethyl)-1,3-dioxolane (1.1 mL, 9.34 mmol), NaHCO₃ (1.57 g 18.6mmol), and NaI (1.40 g, 9.34 mmol) in CH₃CN (20 mL). The reaction wasstirred at 95° C. under argon atmosphere for 15 h as the solution colorturned into brown. The crude product was concentrated under reducedpressure and the residue was purified by reversed-phase columnchromatography to afford 13 (1.45 g, 3.14 mmol, 51%). ¹H NMR (400 MHz,CD₃OD): δ 7.61 (d, 1H, J=7.8 Hz), 7.50 (s, 1H), 6.62 (d, 1H, J=7.8 Hz),4.97 (m, 1H), 4.03-3.93 (m, 4H), 3.71-3.65 (m, 2H), 3.58 (s, 3H),2.19-2.10 (m, 3H), 2.06-2.20 (m, 2H), 1.96-1.90 (m, 2H), 1.56-1.46 (m,2H), 1.43 (s, 3H), 1.05-0.82 (m, 2H). LC-MS (ESI) 440 (M⁺).

1-(2-(1,3-Dioxolan-2-yl)ethyl)-2-((1E,3E)-5-((E)-1-(but-3-en-1-yl)-3,3-dimethyl-5-sulfonatoindolin-2-ylidene)penta-1,3-dien-1-yl)-3-(5-methoxy-5-oxopentyl)-3-methyl-3H-indol-1-ium-5-sulfonate(14)

Acetic anhydride (0.87 mL, 9.21 mmol) was added to a solution of 13(2.10 g, 4.54 mmol),1-(but-3-en-1-yl)-2,3,3-trimethyl-3H-indol-1-ium-5-sulfonate, potassiumsalt (1.51 g, 4.54 mmol), malonaldehyde bis(phenylimine)monohydrochloride (1.21 g, 5.45 mmol), and Et₃N (3.2 mL, 22.7 mmol) inMeOH (23 mL). While stirring at room temperature, additional aceticanhydride (0.87 mL, 9.21) was added every 30 minutes during the first1.5 hours for a total of 3.48 mL (36.8 mmol). During the reaction, thesolution color transitioned from red to green to purple and finally todark blue. After 15 hours of total reaction time, LC/MS analysisrevealed a mixture of the 3 possible cyanine products in anapproximately 2:1:1 ratio of the unsymmetrical desired product to theundesired symmetrical cyanines. A solution of 2:1 diethyl ether/hexanes(180 mL) was added and a precipitate formed, which was collected bycentrifugation. This solid was triturated three times with of 2:1diethyl ether/hexanes and after drying in vacuo the dark blue residuewas purified with reverse-phase chromatography to provide 14 (946 mg,1.09 mmol, 24%) as a dark blue solid. ¹H NMR (400 MHz, CD₃OD): δ8.35-8.25 (m, 2H), 7.89-7.85 (m, 4H), 7.35 (dd, 2H, J=20.1, 8.4 Hz),6.72 (m, 1H), 6.40 (t, 2H, J=12.9 Hz), 5.89 (m, 1H), 5.04-4.95 (m 3H),4.26-4.23 (m, 4H), 3.96-3.93 (m, 2H), 3.85-3.81 (m, 2H), 3.53 (s, 3H),2.62-2.57 (m, 2H), 2.47 (s, 1H), 2.23-2.12 (m, 5H), 1.74 (s, 3H), 1.73(s, 3H), 1.70 (s, 3H), 1.50-1.42 (m, 2H), 0.93 (m, 1H), 0.63 (m, 1H).LC-MS (ESI) 769 (M+).

(7aS,8aR,9aS)-20-(5-Methoxy-5-oxopentyl)-17,17,20-trimethyl-6,7,7a,8a,9,9a,1,11,17,20-decahydrobenzo[2′,3′]indolizino[8′,7′:5,6]pyrano[2,3-g]indolo[2,1-a]isoquinolin-5-ium-2,15-disulfonate(16)

Acrolein dimethyl acetal (1.16 mL, 0.98 mmol) was added to a solution of4 (850 mg, 0.98 mmol), Hoyveyda-Grubbs catalyst 2^(nd) generation (313mg 0.50 mmol) and tetrabutylammonium bromide (946 mg, 2.94 mmol) inanhydrous CH₂Cl₂ (50 mL). The flask was vacuumed and flushed with argonapproximately every 30 min for 5 h. The solution was stirred for anadditional 10 h. A solution of 1:1 diethylether/hexanes (360 mL) wasadded to the blue mixture and the precipitate was collected bycentrifugation. After the second precipitation and centrifugation, thepellet was dried under high vacuum to afford 15 (694 mg, 75% yield) as adark blue solid that was used without further purification. A dark bluesolution of 15 (694 mg, 0.74 mmol) and tetrabutylammonium bromide (714mg, 2.22 mmol) in CH₂Cl₂ (49 mL) was degassed and cooled to −78° C.under argon. BBr₃ solution (8.6 mL, 8.6 mmol, 1.0 M in CH₂Cl₂) was addedslowly and the reaction color transitioned to red-brown. The reactionwas stirred under argon with the temperature at −78° C. for 4 hours, atwhich time the reaction was quenched with the addition of H₂O (40 mL)and immediately returned to a blue color. After warming to roomtemperature, the organic solvent was evaporated under reduced pressure.The remaining blue aqueous solution was purified by reverse-phasechromatography to yield a diastereomeric mixture product (367 mg). Adiastereomeric mixture (367 mg, 0.50 mmol) was dissolved in MeOH (41 mL)and aqueous HCl (0.30 M, 120 mL). This reaction mixture was heated to60° C. for 7 h over which time the color transitioned from dark blue toa green-blue color. After evaporation of solvents, the crude mixture waspurified by reverse-phase chromatography to afford 6 (294 mg, 0.40 mmol,41% yield) as a blue solid. ¹H NMR (400 MHz, d6-DMSO): δ 8.00 (s, 1H),7.93 (s, 1H), 7.82 (d, 1H, J=1.2 Hz), 7.72 (m, 1H), 7.64 (dd, 1H, J=8.2,1.6 Hz), 7.60 (dd, 1H, J=8.2, 1.6 Hz), 7.26 (d, 1H, J=8.2 Hz), 7.16 (dd,1H, J=8.2, 2.0 Hz), 4.56 (dd, 1H, J=11.3, 5.1 Hz), 4.48 (m, 1H),4.30-4.18 (m, 2H), 3.84-3.77 (m, 2H), 3.26 (s, —OMe), 3.45 (s, —OMe),2.70 (m, 1H), 2.46-2.30 (m, 3H), 2.28-2.12 (m, 4H), 1.83 (m, 1H), 1.70(s, 3H), 1.69 (s, 3H), 1.65 (s, 3H), 1.45-1.28 (m, 3H), 0.82 (m, 1H),0.57 (m, 1H). LC-MS (ESI) 735 (M⁺).

5-((7aS,8aR,9aS)-17,17,20-trimethyl-2,15-disulfonato-6,7,7a,8a,9,9a,10,11,17,20-decahydrobenzo[2′,3′]indolizino[8′,7′:5,6]pyrano[2,3-g]indolo[2,1-a]isoquinolin-5-ium-20-yl)pentanoate(17)

Compound 16 (239 mg 0.32 mmol) was dissolved in MeOH (14 mL) and aqueousLiOH (2.0 M, 14 mL). The resulting blue solution was stirred at roomtemperature for 2.5 hours. Saturated aqueous NaHCO₃ (12.0 mL) was addedto quench the reaction. After the removal of MeOH in vacuo, the crudeaqueous mixture was purified by reverse-phase chromatography to afford17 (101 mg, 0.14 mmol, 43% yield) as a blue solid. ¹H NMR (400 MHz,d6-DMSO): δ 8.01 (d, 1H, J=3.9 Hz), 7.92 (s, 1H), 7.82 (d, 1H, J=1.2Hz), 7.72 (m, 1H), 7.63 (dd, 1H, J=8.2, 1.6 Hz), 7.60 (dd, 1H, J=8.2,1.6 Hz), 7.25 (d, 1H, J=8.2 Hz), 7.16 (dd, 1H, J=8.4, 3.7 Hz), 4.56 (m,1H), 4.48 (td, 1H), 4.30-4.19 (m, 2H), 3.84-3.76 (m, 2H), 2.70 (m, 1H),2.42-2.30 (m, 3H), 2.26-2.17 (m, 2H), 2.08-2.01 (m, 2H), 1.84 (m, 1H),1.69 (s, 3H), 1.68 (s, 3H), 1.65 (s, 3H), 1.40-1.30 (m, 3H), 0.83 (m,1H), 0.63 (m, 1H). LC-MS (ESI) 721 (M+).

VII. Representative Embodiments

Certain representative embodiments are disclosed in the followingnumbered clauses.

1. A compound having a chemical structure according to Formula I, or astereoisomer or pharmaceutically acceptable salt thereof:

wherein A is

wherein each “*” designates an attachment point of A; the bondsrepresented by “

” are single or double bonds as needed to satisfy valence requirements;R¹-R⁹ and R¹¹ independently are H, deuterium, alkyl, heteroalkyl,—N(R^(a))₂, sulfonate, alkyl sulfonate, amino, aminoalkyl, —C(O)OR^(a),or a group comprising a conjugatable moiety, a targeting agent, or adrug, where R^(a) is H, deuterium, alkyl or heteroalkyl; R¹⁰ is H,deuterium, O, alkyl, aryl, amino, sulfonate, triflate, —C(O)OR^(b),—OR^(b), —N(R^(b))₂, heteroalkyl, heteroaryl, or a group comprising aconjugatable moiety, a targeting agent, or a drug, where each R^(b)independently is H, deuterium, alkyl, heteroalkyl, aryl, or heteroaryl;and Y¹ and Y² independently are C(R^(c))₂, N(R^(d)), S, O, or Se,wherein each R^(c) independently is H, deuterium, alkyl,—(OCH₂CH₂)_(x)OH where x is an integer ≥2, or a group comprising aconjugatable moiety, a targeting agent, or a drug, and each R^(d)independently is H, deuterium, alkyl, or heteroalkyl.

2. The compound of clause 1, having a chemical structure according toFormula IA, IB, IC, or ID:

3. The compound of clause 1 or clause 2, wherein at least one of R³ andR⁶ is sulfonate, —C(O)OR^(a), or a group comprising a conjugatablemoiety, a targeting agent, or a drug.

4. The compound of any one of clauses 1-3, wherein Y¹ and Y² areC(R^(c))₂ and each R^(c) independently is C₁-C₃ alkyl, —(CH₂)_(n)C(O)R,or H, wherein n is an integer ≥1 and R^(e) is a conjugatable moiety, atargeting agent, or a drug.

5. The compound of clause 4, wherein Y¹ and Y² are C(CH₃)₂.

6. The compound of any one of clauses 1-5, wherein R¹, R², R⁴, R⁵, R⁷,and R⁸ are H.

7. The compound of any one of clauses 1-6, having a chemical structureaccording to Formula II or Formula III:

8. The compound of clause 7, wherein each R^(c) is —CH₃.

9. The compound of clause 7 or clause 8, wherein the compound has achemical structure according to Formula II and R¹-R¹⁰ are H.

10. The compound of clause 7 or clause 8, wherein R¹, R², R⁴, R⁵, andR⁷-R¹¹ are H, and R³ and R⁶ independently are —SO₃ or —CO₂R^(a).

11. The compound of clause 7 or clause 8, wherein the compound has achemical structure according to Formula II, R⁹ and R¹⁰ are H, and atleast one of R³ and R⁶ is a group comprising a conjugatable moiety, atargeting agent, or a drug.

12. The compound of clause 7 or clause 8, wherein the compound has achemical structure according to Formula III, R¹-R⁹ and R¹¹ are H, andR¹⁰ is H, O, triflate, aryl, —OR^(b), or —N(R^(b))₂.

13. The compound of clause 7 or clause 8, wherein the compound has achemical structure according to Formula III, R⁹ and R¹¹ are H, and atleast one of R³, R⁶, and R¹⁰ is a group comprising a conjugatablemoiety, a targeting agent, or a drug.

14. A pharmaceutical composition comprising a compound according to anyone of clauses 1-13 and a pharmaceutically acceptable carrier.

15. A method for making a compound according to clause 1 wherein A is

the method comprising: combining a solution comprising a compoundaccording to Formula IV with 3-buten-1-yl trifluoromethanesulfonate toproduce a compound according to Formula V

combining a solution comprising the compound according to Formula V anda compound according to Formula VI withN-((1E,3Z)-3-(phenylamino)propo-1-en-1-yl)aniline to form a compoundaccording to Formula VII

combining a solution comprising the compound according to Formula VIIwith 3,3-dimethoxy-1-propene in the presence of a ruthenium catalyst toprovide a compound according to Formula VIII

combining the compound according to Formula VIII with (i) a mixture ofCHCl₃ and H₂SO₄ or (ii) BBr₃ in CH₂Cl₂ to provide a compound accordingto Formula IX:

16. The method of clause 15, wherein the ruthenium catalyst is(1,3-bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene)dichloro(o-isopropoxyphenylmethylene)ruthenium.

17. A method for making a compound according to clause 1 wherein A is

the method comprising: combining a solution comprising a compoundaccording to Formula IV with 3-buten-1-yl trifluoromethanesulfonate toproduce a compound according to Formula V

combining a solution comprising a compound according to Formula X with3-buten-1-yl trifluoromethanesulfonate to produce a compound accordingto Formula XI

combining a solution comprising the compound according to Formula V andthe compound according to Formula XI, wherein the compounds according toFormula V and Formula XI may be the same or different, with(1E,4E)-1,5-bis(dimethylamino)-penta-1,4-dien-3-one to produce acompound according to Formula XII

combining a solution comprising the compound according to Formula XIIwith 3,3-dimethoxy-1-propene in the presence of a ruthenium catalyst toprovide a compound according to Formula XIII

andcombining the compound according to Formula XIII with a solution of 1 NHCl in tetrahydrofuran to provide a compound according to Formula XIV

18. The method of clause 17, wherein the ruthenium catalyst is(1,3-bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene)dichloro(o-isopropoxyphenyl-methylene)ruthenium.

19. The method of clause 17, further comprising combining a solutioncomprising the compound according to Formula XIV withtrifluoromethanesulfonic anhydride (Tf₂O) to provide a compoundaccording to Formula XV:

20. The method of clause 19, further comprising combining a solutioncomprising the compound according to Formula XV with R^(g)—C₆H₄—B(OH)₂in the presence of a palladium catalyst to provide a compound accordingto Formula XVI:

where R^(g) is R^(a), —COOR^(a), or —OR^(a), where R^(a) is H,deuterium, alkyl or heteroalkyl.

21. The method of clause 19, further comprising combining a solutioncomprising the compound according to Formula XV with an amine having aformula NH(R²⁰)(R²¹) to provide a compound according to formula XVII:

where R²⁰ and R²¹ independently are H, deuterium, alkyl, heteroalkyl,aryl or heteroaryl.

22. The method of clause 21, wherein (i) R²⁰ is —(CR^(h) ₂)_(n)-CH₂OHwhere each R^(h) independently is H, deuterium, halo, alkyl, or aryl,and n is 1, 2, 3, or 4, and (ii) R²¹ is H, deuterium, alkyl,heteroalkyl, aryl, or heteroaryl, the method further comprisingcombining a solution comprising the compound according to Formula XVIIwith a compound comprising an electrophilic group R²² under basicconditions to provide a compound according to Formula XVIII:

23. A method for using a compound according to any one of clauses 1-13wherein at least one of R¹-R¹¹ comprises a targeting agent, the methodcomprising: combining the compound with a sample comprising a targetcapable of binding with the targeting agent under conditions effectiveto provide binding of the targeting agent and the target; and imagingthe target by visualizing the compound bound to the target.

24. The method of clause 23, wherein visualizing the compound comprises:irradiating the sample with targeted application of a quantity of lighthaving a wavelength in the visible or near-infrared range and a selectedintensity, wherein the quantity of light is sufficient to producefluorescence of the compound; and detecting any fluorescence emitted bythe compound.

25. The method of clause 23 or clause 24, wherein combining the compoundwith the sample is performed in vitro, ex vivo, or in vivo.

26. The method of any one of clauses 23-25, further comprising combiningthe compound with a reducing agent prior to imaging the target.

27. The method of any one of clauses 23-26, wherein the sample is atissue sample, a biological fluid, or a target area within a subject.

28. The method of clause 27, wherein the sample is a target area withina subject, the method further comprising: administering the compound, ora pharmaceutical composition comprising the compound to the subject;subsequently irradiating the compound by targeted application of thequantity of light to a targeted portion of the subject; and detectingany fluorescence from the compound in the targeted portion of thesubject.

29. The method of clause 28, wherein the target area is a tumor site andthe targeted portion of the subject includes the tumor site, the methodfurther comprising excising at least a portion of the tumor from thesubject after detecting the fluorescence in the targeted portion of thesubject.

30. A method for detecting reactive oxygen species, the methodcomprising: combining a compound according to any one of clauses 1-13with a reducing agent to provide a reduced compound; contacting a samplewith the reduced compound, whereby the reduced compound is oxidized toregenerate the compound according to any one of clauses 1-13 if reactiveoxygen species (ROS) are present in the sample; irradiating the samplewith a quantity of light having a wavelength in the visible ornear-infrared range and a selected intensity, wherein the quantity oflight is sufficient to produce fluorescence if the reduced compound hasbeen oxidized by the ROS to regenerate the compound according to any oneof clauses 1-13; and detecting any fluorescence emitted by the compoundaccording to any one of clauses 1-13, wherein fluorescence indicates thepresence of ROS in the sample.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1. A compound having a chemical structure according to Formula I, or astereoisomer or pharmaceutically acceptable salt thereof:

wherein A is

wherein each “*” designates an attachment point of A; the bondsrepresented by “

” are single or double bonds as needed to satisfy valence requirements;R¹-R⁹ and R¹¹ independently are H, sulfonate, —N(R^(a))₂, deuterium,alkyl, heteroalkyl, alkyl sulfonate, aminoalkyl, —C(O)OR^(a), trityl, ora group comprising a conjugatable moiety, a targeting agent, or a drug,where each R^(a) independently is H, deuterium, alkyl, or heteroalkyl;R¹⁰ is H, deuterium, O, alkyl, aryl, amino, sulfonate, triflate,—C(O)OR^(b), —OR^(b), —N(R^(b))₂, heteroalkyl, heteroaryl, trityl, or agroup comprising a conjugatable moiety, a targeting agent, or a drug,where each R^(b) independently is H, deuterium, alkyl, heteroalkyl,aryl, or heteroaryl; and Y¹ and Y² independently are C(R^(c))₂,N(R^(d)), S, O, or Se, wherein each R^(c) independently is alkyl, H,deuterium, —(OCH₂CH₂)_(x)OH where x is an integer ≥2, trityl, or a groupcomprising a conjugatable moiety, a targeting agent, or a drug, and eachR^(d) independently is H, deuterium, alkyl, or heteroalkyl.
 2. Thecompound of claim 1, wherein at least one of R³ and R⁶ is sulfonate,—C(O)OR^(a), or a group comprising a conjugatable moiety, a targetingagent, or a drug.
 3. The compound of claim 1, wherein Y¹ and Y² areC(R^(c))₂ and each R^(c) independently is C₁-C₃ alkyl,—(CH₂)_(n)C(O)R^(e), or H, wherein n is an integer ≥1 and R^(e) is aconjugatable moiety, a targeting agent, or a drug.
 4. The compound ofclaim 3, wherein: Y¹ and Y² are C(CH₃)₂; or R³ and R⁶ are sulfonate, andone R^(c) is —(CH₂)_(n)C(O)R^(e).
 5. The compound of claim 1, whereinR¹, R², R⁴, R⁵, R⁷, and R⁸ are H.
 6. The compound of claim 1, whereinthe conjugatable moiety is

where y is an integer ≥1, or a phosphoramidite group.
 7. The compound ofclaim 1, having a chemical structure according to Formula II or FormulaIII:


8. The compound of claim 7, wherein: each R^(c) is —CH₃; R³ and R⁶ aresulfonate; one R^(c) is a group comprising a conjugatable moiety, atargeting agent, or a drug; or R³ and R⁶ are sulfonate, and one R^(c) isa group comprising a conjugatable moiety, a targeting agent, or a drug.9. The compound of claim 7, wherein: the compound has a chemicalstructure according to Formula II and R¹-R¹⁰ are H; the compound has achemical structure according to Formula II, R⁹ and R¹⁰ are H, and atleast one of R³ and R⁶ is a group comprising a conjugatable moiety, atargeting agent, or a drug; the compound has a chemical structureaccording to Formula III, R¹-R⁹ and R¹¹ are H, and R¹⁰ is H, O,triflate, aryl, —OR^(b), or —N(R^(b))₂; or the compound has a chemicalstructure according to Formula III, R⁹ and R¹¹ are H, and at least oneof R³, R⁶, and R¹⁰ is a group comprising a conjugatable moiety, atargeting agent, or a drug.
 10. The compound of claim 7, wherein R¹, R²,R⁴, R⁵, and R⁷-R¹¹ are H, and R³ and R⁶ independently are —SO₃ or—CO₂R^(a).
 11. A pharmaceutical composition comprising a compoundaccording to claim 1 and a pharmaceutically acceptable carrier.
 12. Amethod for making a compound according to claim 1 wherein A is

the method comprising: combining a solution comprising a compoundaccording to Formula IV with 3-buten-1-yl trifluoromethanesulfonate toproduce a compound according to Formula V

combining a solution comprising the compound according to Formula V anda compound according to Formula VI withN-((1E,3Z)-3-(phenylamino)propo-1-en-1-yl)aniline orN-((1E,3E)-3-(phenylimino)prop-1-en-1-yl)aniline to form a compoundaccording to Formula VII

combining a solution comprising the compound according to Formula VIIwith 3,3-dimethoxy-1-propene in the presence of a ruthenium catalyst toprovide a compound according to Formula VIII

and combining the compound according to Formula VIII with (i) acidifiedCHCl₃ or (ii) BBr₃ in CH₂Cl₂ to provide a compound according to FormulaIX:


13. A method for making a compound according to claim 1 wherein A is

the method comprising: combining a solution comprising a compoundaccording to Formula IV with 3-buten-1-yl trifluoromethanesulfonate toproduce a compound according to Formula V

combining a solution comprising a compound according to Formula X with3-buten-1-yl trifluoromethanesulfonate to produce a compound accordingto Formula XI

combining a solution comprising the compound according to Formula V andthe compound according to Formula XI, wherein the compounds according toFormula V and Formula XI may be the same or different, with(1E,4E)-1,5-bis(dimethylamino)-penta-1,4-dien-3-one to produce acompound according to Formula XII

combining a solution comprising the compound according to Formula XIIwith 3,3-dimethoxy-1-propene in the presence of a ruthenium catalyst toprovide a compound according to Formula XIII

and combining the compound according to Formula XIII with a solution ofacidified tetrahydrofuran to provide a compound according to Formula XIV


14. The method of claim 13, further comprising combining a solutioncomprising the compound according to Formula XIV withtrifluoromethanesulfonic anhydride (Tf₂O) to provide a compoundaccording to Formula XV:


15. The method of claim 14, further comprising: combining a solutioncomprising the compound according to Formula XV with R^(g)—C₆H₄—B(OH)₂in the presence of a palladium catalyst to provide a compound accordingto Formula XVI

where R^(g) is R^(a), —COOR^(a), or —OR^(a), where R^(a) is H,deuterium, alkyl or heteroalkyl; or combining a solution comprising thecompound according to Formula XV with an amine having a formulaNH(R²⁰)(R²¹) to provide a compound according to Formula XVII

where R²⁰ and R²¹ independently are H, deuterium, alkyl, heteroalkyl,aryl or heteroaryl.
 16. A method for using a compound according to claim1 wherein at least one of R¹-R¹¹ comprises a targeting agent, the methodcomprising: combining the compound with a sample comprising a targetcapable of binding with the targeting agent under conditions effectiveto provide binding of the targeting agent and the target; and imagingthe target by visualizing the compound bound to the target, preferablywherein visualizing the compound comprises irradiating the sample withtargeted application of a quantity of light having a wavelength in thevisible or near-infrared range and a selected intensity, wherein thequantity of light is sufficient to produce fluorescence of the compound,and detecting any fluorescence emitted by the compound.
 17. The methodof claim 16, further comprising combining the compound with a reducingagent prior to imaging the target.
 18. The method of claim 16, whereinthe sample is a target area within a subject, the method furthercomprising: administering the compound, or a pharmaceutical compositioncomprising the compound to the subject; subsequently irradiating thecompound by targeted application of the quantity of light to a targetedportion of the subject; and detecting any fluorescence from the compoundin the targeted portion of the subject.
 19. The method of claim 18,wherein the target area is a tumor site and the targeted portion of thesubject includes the tumor site, the method further comprising excisingat least a portion of the tumor from the subject after detecting thefluorescence in the targeted portion of the subject.
 20. A method fordetecting reactive oxygen species, the method comprising: combining acompound according to claim 1 with a reducing agent to provide a reducedcompound; contacting a sample with the reduced compound, whereby thereduced compound is oxidized to regenerate the compound according toclaim 1 if reactive oxygen species (ROS) are present in the sample;irradiating the sample with a quantity of light having a wavelength inthe visible or near-infrared range and a selected intensity, wherein thequantity of light is sufficient to produce fluorescence if the reducedcompound has been oxidized by the ROS to regenerate the compoundaccording to claim 1; and detecting any fluorescence emitted by thecompound according to claim 1, wherein fluorescence indicates thepresence of ROS in the sample.